Polyamides for nucleic acid delivery

ABSTRACT

The present invention provides a new class of non-viral transduction vectors that can be used for both in vivo and in vitro applications. The present invention also provides a gene transfer vector that has comparable efficiency to a viral vector without the potential for a life-threatening immune response. Complexes according to the invention or portions thereof, can comprise a cellular delivery molecule or agent that can facilitate the translocation of the complex or portion thereof into cells. In some embodiments, cellular delivery molecules for use in the present invention may comprise one or more polymers of the present invention, e.g., polyamides, dendritic macromolecules and carbohydrate-containing degradable polyesters.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/531,399, filed Dec. 19, 2003 and U.S.Provisional Patent Application Ser. No. 60/574,131, filed May 25, 2004,which applications are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention is in the fields of molecular biology,biochemistry and pharmaceuticals. In general, the invention providescompositions for the cellular delivery of nucleic acids, polypeptidesand/or molecular complexes comprising nucleic acids and polypeptides,and methods of making and using such compositions. The present inventionprovides a new class of non-viral transduction vectors that can be usedfor both in vivo and in vitro applications. The present inventionprovides for unique polycationic polymers that can associate with manysuitable bioactive molecules, including proteins and other compoundsthat poses multiple cationic sites. The polymer can act as a deliveryvehicle for the associated bioactive molecule, in vivo or in vitro, tothe cells of interest for the bioactive molecule. In one embodiment, thepresent invention provides for a new series of polyamides for use asgene delivery agents. Also disclosed are methods of using the polymersto bind products, e.g., oligonucliotides, and facilitate cellularuptake. In one embodiment, the invention provides for the in vitrodelivery of plasmid DNA into cells. The present also provides for theuse of these polymers for the delivery of a nucleic acid is biologicallyactive into a cell.

BACKGROUND OF THE INVENTION

Nucleic acids show great promise as new therapeutics to treat bothacquired and inherited diseases. One of the greatest challenges with thesuccessful application of nucleic acid drugs is the development of anefficacious delivery method.1 Delivery systems are needed to compactgenetic material into nanostructures that can be taken up by cells,protect nucleic acids from enzymatic damage during cellular transport,and provide the possibility of targeting the delivery to specific celltypes.2 Viral vectors are still the most effective and commonly usedmethod of DNA transport even though many problems with this deliverymethod have been revealed.2,3

Polymer-mediated gene delivery has recently emerged as a viablealternative to viral-based transduction systems since polymers may notinduce immune and inflammatory responses, have a lower cost ofsynthesis, and have a large nucleic acid loading capacity.1,2 Severalstudies have shown that polycations bind DNA electrostatically and formpolyplexes (polymer+DNA complexes) that are endocytosed by many celltypes and deliver DNA with varying degrees of delivery efficiency andtoxicity.4,5 Although synthetic delivery systems show great promise,difficulties with polymer toxicity and low delivery efficiency havehampered clinical application of these vectors.1,2 For example,polyethylenimine (PEI), a polymer of ethylenediamine, exhibits efficientgene delivery but is also very cytotoxic.6 Conversely, chitosan, apolymer of glucosamine, is completely nontoxic yet reveals low deliveryefficiency in many cell lines.7 Progress towards rationally-designedsynthetic delivery systems has also been stalled by a lack ofunderstanding of the fundamental polymer structure-biological propertyrelationships that exist for synthetic delivery vehicles.4,5

Drug delivery is an important field for both clinical applications andresearch. Some biological systems possess unique delivery challenges.

In recent years gene therapy has received a greater amount of attentionin academic and scientific circles. The potential for gene therapy forpharmaceutical, commercial, and clinical applications is tremendous.Gene transfection, the addition of a gene to a cell, is a criticalcomponent of gene therapy.

Presently there are several approaches to gene transfection. Theseinclude the use of viral based vectors (e.g., retroviruses,adenoviruses, and adeno-associated viruses) (Drumm, M. L. et al., Cell62:1227-1233 (1990); Rosenfeld, M. A. et al., Cell 68:143-155 (1992);and Muzyczka, N., Curr. Top. Micro. Immuno. 158:97-129 (1992)), chargeassociating the DNA with an asialorosomucoid/poly L-lysine complex(Wilson, J. M. et al., (1992)), Charge associating the DNA with cationicliposomes (Brigham, K L. et al., (1993)) and the use of cationicliposomes in association with a poly-L-lysine antibody complex(Trubetskoy, V. S. et al., Biochem. Biophys. Acta 1131:311-313 (1993)).

Viral vectors have exhibited the highest levels of transfectionefficiency to date for nucleic acids. Viral vectors have beenparticularly effective in in vivo systems, where other transfectionsystems have fallen short. Viral vectors do have a tremendous downside,namely the potential to illicit a potentially life-threatening immuneresponse. (Kingman, Bioworld Int., 1(20):l (1996)). This happens becausethe viral carrier actually infects the cell as part of the method oftransfection.

Although non-viral based transfection systems have not exhibited theefficiency of viral vectors, they are still receiving significantscientific attention because of their probable increased safety for invivo systems. This has also led to increased attention for in vitrosystems as well. Synthetic cationic molecules have been reported to“coat” the nucleic acid through interactions on the cationic sites ofthe transfection reagent and the anionic sites on the nucleic acid. Thepositively charged coating reportedly interacts with the negativelycharged cell membrane to facilitate the passage of the nucleic acid intothe cytoplasm via non-specific endocytosis. (Schofield, Brit.Microencapsulated. Bull., 51(1):56-71 (1995)).

Past attempts at nucleic acid transfection have also experienceddifficulty with DNA precipitating out of solution. The problem isespecially acute in in vivo applications where typically higherconcentrations of DNA are present. These higher concentrations createsolubility problems for the DNA/carrier systems. DNA precipitation canbe avoided by increasing the concentration of mono- and polyvalentcations. In the past this had partly solved the DNA solubility problem,but it also increased the toxic effects upon the transfected cells.

SUMMARY OF THE INVENTION

The present invention provides a new class of non-viral transductionvectors that can be used for both in vivo and in vitro applications. Inparticular, these vectors can be used for gene transfer applications.These new gene transduction vectors can achieve transfer efficienciesfar greater to commercially available polymeric and liposomal genetransfer vectors while maintaining little or no toxicity in vitro. Theirlow in vitro toxicity makes them ideal candidates for in vivo use. Thepresent invention also provides a gene transfer vector that hascomparable efficiency to a viral vector without the potential for alife-threatening immune response.

Furthermore, the unique polycationic structure of these polymersassociates with many suitable biologically active molecule, includingoligonucleotides and polypeptides and other compounds that posesmultiple cationic sites. The polymer can act as a delivery vehicle forthe associated biologically active molecule, in vivo or in vitro, to thecells of interest for the biologically active molecule.

In one embodiment, the invention encompasses a method of delivering abiologically active molecule to a cell, comprising contacting the cellwith (a) a biologically active molecule and (b) a cellular deliverypolymer.

In one embodiment, the present invention also provides for compositionsand non-covalent complexes comprising one or more polymers of thepresent invention, e.g., polyamides, dendritic macromolecules (polymerscomprising an oligoamine shell and a cyclodextrin core), andcarbohydrate-containing degradable polyesters, and at least one nucleicacid molecule (e.g., one or more oligonucleotides) or at least onepolypeptide or both. The invention also provides compositions comprisingsuch complexes.

Complexes according to the invention or portions thereof, can comprise acellular delivery molecule or agent that can facilitate thetranslocation of the complex or portion thereof into cells. In someembodiments, cellular delivery molecules for use in the presentinvention may comprise one or more one or more polymers of the presentinvention, e.g., polyamides, dendritic macromolecules (polymerscomprising an oligoamine shell and a cyclodextrin core), andcarbohydrate-containing degradable polyesters.

In some embodiments, a cell, tissue, organ or organism may be contactedwith a complex of the invention. Preferably, the complex is taken up bythe cell or by one or more cells of the tissue, organ or organism.

In another exemplary and non-limiting embodiment of the invention,compositions comprising complexes between cellular delivery polymers andoligonucleotides are formed and can be applied to cultured mammaliancells. The complex may also comprise a combination of labeled andnonlabeled nucleic acid and or peptide. These complexes allow mediationof an activity associated with the oligonucleotide, which, by way ofnon-limiting example, can be a gene-containing oligonucleotide, anantisense oligonucleotide, an aptamer, a short interfering RNA (siRNA),a short hairpin RNA (shRNA), a small temporally regulated RNA (stRNA),and the like. In some embodiments, oligonucleotides are preferred.

In other specific embodiments, the biologically active molecule and/orcell delivery agent is covalently labeled with a fluorophores(fluorescent moiety), for example with fluorescein or a derivative offluorescein.

In another embodiment, the compositions may comprise one or morefluorescent molecules or moieties, which may be the same or different,and may be covalently attached to one or more polypeptides and/ornucleic acid molecules in the complexes of the invention. Alternatively,or in addition, complexes of the invention may comprise one or more“free” fluorescent molecule (i.e., one or more fluorescent moleculesthat are not covalently attached to either the polypeptide or theoligonucleotide but may still be associated with the complex). One ormore of the compounds of the compositions or complexes can be abiologically active molecule.

Kits according to the invention may further comprise one or moretransfection agents, one or more cells, one or more nucleic acids, oneor more set of instructions, and one or more biologically activemolecules.

Other additional kit components include without limitation: additionalnucleic acids, such as oligonucleotides, iRNA molecules, plasmids, etc.;one or more recombinases, including without limitation site-specificrecombinases; one or more recombination proteins; and/or one or morecells. In some embodiments, the cells are competent for transfection ortransformation.

In other embodiments, the invention provides a complex comprising a celldelivery polymer and a biologically active agent that is desirably takenup by cells, wherein the cell delivery polymer or biologically activeagent comprises a fluorescent moiety.

The nucleic acid of the complexes and other embodiments of the inventioncan comprise from 5 bases to about 200 kilobases. Any type of nucleicacid may be used, including by way of non-limiting example mRNA, tmRNA,tRNA, rRNA, siRNA, shRNA, PNA, ssRNA, dsRNA, ssDNA, dsDNA, DNA:RNAhybrid molecules, plasmids, artificial chromosomes, gene therapyconstructs, cDNA, PCR products, restriction fragments, ribozymes,antisense constructs, and combinations thereof. Reviews of tmRNA includeMuto A, Ushida C, Himeno H. A bacterial RNA that functions as both atRNA and an mRNA. Trends Biochem Sci. 1998 January; 23(1):25-9; andWithey J H, Friedman D I. The biological roles of trans-translation.Curr Opin Microbiol. 2002 April; 5(2):154-9). The nucleic acid maycomprise one or more chemical modifications.

A complex according to the invention may further comprise one or moretransfection agents, one or more recombinases and, additionally oralternatively, one or more recombination proteins.

A nucleic acid used in the invention includes, in some embodiments, asequence that encodes a protein or a portion thereof. In someembodiments, a cellular nucleic acid encoding the protein, or a portionthereof, is desirably replaced by the sequence in one form of genetherapy. Additionally or alternatively, the protein is expressed in thecell. The protein may be exogenous or endogenous. In the latter case,the cells to be transfected may comprise a non-functional form of theprotein.

A composition of the invention may be a pharmaceutical composition. Incertain embodiments, the biologically active molecule is one or more ofthe nucleic acids that has a biological activity, including but notlimited to therapeutic activity. By way of non-limiting example,biologically active nucleic acids are selected from the group consistingof mRNA, tmRNA, tRNA, rRNA, siRNA, shRNA, PNA, ssRNA, dsRNA, ssDNA,dsDNA, DNA:RNA hybrid molecules, plasmids, artificial chromosomes, genetherapy constructs, cDNA, PCR products, restriction fragments,ribozymes, antisense constructs, and combinations thereof.

Additionally or alternatively, polypeptide of the complex isbiologically active. A biologically active polypeptide may be atherapeutic protein. By way of non-limiting example, bioactive proteinsinclude antibodies or antibody fragments, hormones, enzymes,transcription factors, growth factors, and the like.

The invention further provides a method of providing gene therapy to anindividual in need thereof, of treating an individual suffering from adisease or disorder, the method comprising contacting the individual, orcells therefrom, with one or more complexes, compositions and/orpharmaceutical compositions of the invention.

The invention further provides a method of testing a cellular responseto a test compound, the method comprising: (a) contacting a first cellwith, in any order or combination, a biologically active molecule and acellular delivery molecule; (b) contacting a second cell with, in anyorder or combination, a second biologically active molecule and thecellular delivery molecule; (c) contacting the cells with the testcompound, before (a); during (a) or (b); between (a) and (b); and,additionally or alternatively, after (b); (d) measuring and comparing atleast one parameter of from the first cell with the signal from thesecond cell. In certain embodiments, one or more of the cells compriseone or more reporter genes that generate a detectable signal orinterfere with the production of a detectable signal.

In one embodiment, the present invention provides for a new series ofpolyamides for use as gene delivery agents. These polymers bindproducts, e.g., oligonucliotides, and facilitate cellular uptake. In oneembodiment, the invention provides for the in vitro delivery of plasmidDNA into cells. In one embodiment, the invention provides for the invivo delivery of plasmid DNA into cells.

In one embodiment, the present invention relates to the use ofpolyamides for delivering nucleic acids into a cell. In one embodiment,the nucleic acid is an oligonucleotide. In another embodiment, theoligonucleotide contains from about 10 to about 1000 nucleotides. Inanother embodiment, the oligonucleotide is an antisense oligonucleotideor oligodeoxynucleotide. In another embodiment, the oligonucleotide isan oligonucleotide, an antisense oligonucleotide residue oroligodeoxynucleotide residue.

In another embodiment, the nucleic acid is selected from the groupconsisting of antisense constructs, antisense polynucleotide, artificialchromosomes, cDNA, concatemers, concatemeric decoy oligonucleotides, CpGoligomers, cyclic oligonucleotides, decoy oligonucleotides, DNA:RNAhybrid molecules, dsDNA, dsRNA, gene therapy constructs, LNA,morpholinos, mRNA, oligonucleotides and oligodeoxynucleotides withphosphorodiester backbones or phosphorothioate backbones, PCR products,plasmids, PNA, restriction fragments, ribozyme, RNA, RNAi, RNAi inducingpolynucleotide, rRNA, shRNA, siRNA, spiegelmers, ssDNA, ssRNA, tmRNA,transgenes, tricyclo-DNA, triple helices, tRNA, and combinationsthereof.

In another embodiment, the present invention provides for the use forpolyamides to deliver a concatemer to a cell. In another embodiment, thepresent invention provides for the use for polyamides to deliver aconcatemerized double-stranded oligonucleotide molecules (CODN) fortranscription factor decoys. In one embodiment, the concatemers consistof a variable number of end-to-end repeated copies of a short (more than5, 10, 15, 20, 2, 3035, 40, 45, 50, 75, 100, or more bp but generallyless than about 3 kb) dsDNA containing a sequence or sequences that actas transcription factor decoys.

The use of the concatemers provides one or more of the followingbenefits: a) increased half-life of the nucleotide within the cell; b)increased efficacy of each single molecule, since each contains multiplecopies of the specific decoy; c) the molar amount of decoy can betitrated to achieve a specific degree of transcription factor blockade;d) CODNs can be designed to block subsets of transcription factorbinding sites that may underlie biological variation in transcriptionfactor response; e) a combinatorial blockade, since each CODN can bindmultiple transcription factors, where use of concatemers allows fordelivery of decoys for 2 or more transcription factors to be done in aprecisely controlled manner. This latter point is relevant to twoimportant issues. First, to any use requiring titration of transcriptionfactor blockade, especially of one transcription factor relative toanother. For instance, if one wishes to completely block factor X andblock factor Y only 25%, this can be done by empirically determining theratio of the decoy for X and Y required and assembling the CODN to thisrequirement. Second, to the fact that transcription factors often acttogether to activate discrete subsets of genes. For instance, NF-kB andAP-1 each act primarily on a certain subset of promoters. There ishowever, a common subset that requires the cooperative binding of bothtranscription factors to nearby sites on the promoter to properlyactivate gene expression. The concatemer allows blocking of these geneswith relative specificity by titrating the decoys for the twotranscription factors, or by designing a unique CODN to the specificcombination of NF-kB and AP-1 binding sites found in the specificpromoter.

In another embodiment, the present invention provides for the use of thepolymers for covalent addition of targeting peptides, receptor bindingpeptides/protein domains and antibody fragments that may be used totarget the CODN/polymer complexes to a specific cell type; thus theagent can be made organ, tissue and/or cell-type specific.

In another embodiment, the present invention provides for usingpolyamides for targeting peptides and/or antibodies for specific stressand/or drug induced cellular receptors. In one embodiment, thepolyamides target the CODN/polymer complexes to ischemic, inflamed orcancerous tissues.

In another embodiment, the present invention provides for using linkerpeptides containing the sequence recognized by the TNF-alpha convertingenzyme (TACE) or another exopeptidase or endopeptidase in order to allowthe agent to deliver the CODN/polymer complex to the cell and thencleave off the targeting peptide.

In another embodiment, the present invention provides for using thepolyamides to deliver intact genes (transgenes), plasmids, RNAi, siRNA,morpholinos or other kinds of RNA, proteins and polynucleotides. In oneembodiment, the genes incorporate tissue-specific promoters,controllable promoters, promoters that may be silenced by specificCODN/polymer combinations and may constitute two- and three-unit systemsfor gene expression, control and DNA transposition (i.e. insertion,excision and targeting of transgenes and other DNA molecules).

In another embodiment, the present invention provides for use of thepolyamides in vitro or in vivo, in isolated cells or intact animals inwhich specific blockade of transcription factors or delivery of DNA orother biological effector is desirable. In one embodiment, this includesuse as a research tool, including studies of specific genes and studiesto identify specific genes regulated by the transcription factorstargeted (relates to development of specific CODN/polymer complexes andrelated gene marker mouse lines described below). For clinical use, thiswould include, but is not limited to delivery of transcription factordecoys (e.g. CODNs) that block transcription factors implicated indisease, response to surgery and/or trauma, developmental defects,aging, toxic exposure, etc.

In another embodiment, the present invention provides for usingpolyamides for NF-kB-specific CODN delivery in the treatment ofmyocardial ischemia/reperfusion and myocardial infarction, heart failureand hypertrophy, cardioprotection, stroke, neuroprotection, sepsis,arthritis, asthma, heritable inflammatory disorders, cancer, heritableimmune dysfunctions, inflammatory processes, whether caused by diseaseor injury or infection, oxidative stress to any organ whether caused bydisease, surgery or injury. In another embodiment, the present inventionprovides for using polyamides for delivery of CODN/polymer complexes todelineate in animal models, specific situations in which NF-kB or othertranscription factors contribute to injury, dysfunction, morbidity ormortality, determine whether blockade is beneficial in animals and thentranslating this to the clinic.

In another embodiment, the present invention provides for transgenicmice expressing marker genes (lacZ and/or GFP variants) under thecontrol of promoter elements that are primarily controlled by specifictranscription factors. In one embodiment, the mice are providedseparately or as a kit including specific CODN/polymer complexes and thematching mouse, which serves to identify the cells in which the markeractivation (experimentally activated) is blocked by the CODN. In anotherembodiment, there are transgenic mice with marker genes that aretranscriptionally turned on, which can be specifically turned off usingCODN/polymer complexes.

In another embodiment, the present invention provides for bi-transgenic(or multiple transgenic) systems designed to utilize the CODN/polymercomplexes to regulate gene expression (up, down, on or off) or tomediate gene transposition (insertion, excision or moving in thegenome). In one embodiment, transgene A may express a gene of interestunder control of a promoter that is inducible by NF-κB or by a yeast orbacterial transcription factor (think tetR or Gal4). In one embodiment,the gene would be on after an NF-kB-inducing stimulus, or constitutivelyon in a tissue expressing the specific transcription factor (we aremaking mice for NF-kB activation; mice for gal4 and tetR already exist)and the gene could be turned off by simply providing the CODN/polymercomplexes for the specific transcription factor (CODN-OFF). In anotherembodiment, the animals are continuously delivered CODN/polymercomplexes and then the CODN/polymer complexes is withdrawn to turn thegene on. Other versions could have the gene off, due to expression inthe same cells of a transcriptional repressor (has been described fortet), and the repression reversed by adding CODN/polymer complexes,allowing expression to turn on (CODN-ON).

Another embodiment provides for the delivery of transgenes that may beincorporated into the genome via retroviruses, transposons orretrotransposons. In one embodiment, the delivery is for long-term geneexpression or genetic engineering in vitro, in vivo, in isolated cellsor in whole animals or in the clinic. In another embodiment, germ cellsare targeted using compositions of the present invention to achieveheritable transgenic lines of animals without having to domicroinjection (optionally using a bi-transgenic system).

In another embodiment, the present invention provides for polymersdesigned for variable release/biodegradation; some may be designed,selected for quick degradation/release of CODN, others for longhalf-life (the CODN may be active whether or not it is released by thepolymers, so we should safeguard the concept that long-lasting bindingof the DNA by polymers, may be a way to prolong activity).

In another embodiment, the present invention provides for the deliveryof one or more imaging agents for real-time and still imaging within acell or tissue.

In another embodiment, the present invention provides for usingpolyamides for delivery of transcription factor decoys (including, butnot limited to NF-kB), to block signaling and gene expression associatedwith pathogenesis.

In another embodiment, the present invention provides for usingpolyamides for delivery of linear duplications or chains of these decoys(i.e., concatemers), such that each strand contains a number of decoytranscription factor binding sites including more than 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, or more. In another embodiment, the presentinvention provides for using polyamides for delivery of decoys havingfor multiple transcription factors into one of these strands, such thatit can affect blockade of 2, 3, 4, 5, 6, or more transcription factorssimultaneously in a cell. In another embodiment, the present inventionprovides for using polyamides for delivery of these strands, or thestrands contained in a plasmid or other DNA vector (can include phage,viral or other DNA) to bind to the polymers to deliver the strands tothe cytoplasm of the cell, to effect transcription factor blockade.

The decoys may be any transcription factors, including, but not limitedto, NF-kB, AP-1, ATF2, ATF3, SP1 and others. This is all based on thenovel concept, supported by data in our lab, that blocking key signalingmolecules simultaneously can have additive or even synergistictherapeutic effects, particularly when the molecules chosen are keysignaling hubs. In signaling, transcription factors participate byactivating or turning down gene expression. In another embodiment, thepresent invention provides for using polyamides for treatment of MI byblocking NF-kB using decoys to iNOS and Cox2. In another embodiment, thepresent invention provides for using polyamides for delivery of decoysto metallothionein and heat shock protein 70.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

Polymeric materials are described for the delivery of therapeutic DNA.The use of synthetic delivery agents has many advantages over viraldelivery vectors for several reasons such as they may not induce immuneand inflammatory responses and thus can be used repeatedly in clinicaladministration. In addition, synthetic vectors have a lower cost, areeasier to manufacture on a larger scale, and they have the ability tocarry an unlimited amount of genetic information.

Polycations self assemble with DNA through electrostatic interactionsand compact DNA into small complexes that have been termed polyplexes.The formation of polyplexes usually occurs at a N/P ratio [the ratio ofpolymer nitrogens (N)/phosphate groups (P) on DNA] greater than one.Polyplexes can be taken up by cells through the endocytotic pathway.Without wishing to be limited by theory in any way, it is believed thatafter uptake, some of the polyplexes are able to escape the endosomesand are transported into the nucleus (most likely during cell division)where the delivered gene is transcribed. The polyamides of the presentinvention are created from comonomers (x=1, L-tartrate and x=2,galactarate).

In one embodiment, the present invention provides for a series ofpolymers for use to probe the structure-property relationships forsynthetic vectors. Described are a series of polyamides that vary in theamount of the hydroxyl and secondary amine groups along the polymericbackbone.

In addition, we have systematically increased the number of secondaryamines between the carbohydrates, in order to elucidate how the numberof basic groups within a polymer repeat unit facilitates efficientnucleic acid binding, condensation, and intracellular gene delivery. Tothis end, we have selected a series of co-monomers that has allowed usto design in these chemical characteristics to yield both biotolerable(i.e., a nontoxic) and highly efficient delivery vehicle.

Polycations self-assemble with biologically active, molecules, and inparticular nucleic acids and peptides, through electrostaticinteractions and they compact DNA into small complexes that have beentermed polyplexes. This has previously been disclosed in U.S. Pat. No.5,948,878, Burgess et al., which is herein incorporated by reference inits entirety. The formation of the polyplexes usually occurs at a N/Pratio [the ratio of polymer nitrogens (N) to phosphate groups (P) andthe DNA] greater than one. Polyplexes can be taken be taken up by thecell by through the endocytic pathway. After endocytosis the polyplexesare able to escape the endosomes and are able to enter the nucleus wherethe delivered gene is transcribed and translated into the desiredprotein. The polymers can be used to deliver any type, length, sequence,and shape of nucleic acid to any cellular destination.

The polymer structure plays a large role in the binding affinity of DNAand the compaction of DNA into polyplexes. Also, the polymer chemistrydictates the efficiency of polyplex cellular uptake and endosomalrelease within the cytoplasm. Furthermore, the polymer structure hasbeen shown to significantly affect both the delivery efficiency andtoxicity that is observed during gene transport.

The Classes of Polymers

Note: The term “polymers” is used throughout the application and thisrefers to the classes of polymers used for polyplex formation.Therefore, the term polymer includes poly(hydroxylamidoamine), dendriticmacromolecules, and also carbohydrate-containing biodegradablepolyesters.

1) Poly(hydroxylamidoamine)s

These polyamides, including but not limited to poly(glycoamidoamine)s,(any carbohydrate) and poly(L-tartaramidoamine)s, may be prepared bycondensation of an appropriately substituted diester or othersubstitutions that react with amines such as acid chlorides, carboxylicacids, lactones, anhydrides, etc. and an appropriately substituteddiamine comonomer.

Diesters include, but are not limited to, those shown below, theirstereoisomers, mixtures of isomers, and also includeD-Mannaro-1,4-:6,3-dilactone, dimethyl-meso-galctarate, esterifiedglucaric acid, dimethyl D-glucarate (linear and closed ring forms of allstereoisomers), esters of methyl citric acid, methyltartronic acid,methyl D-arabinaric acid, and esters of xylaric acid and methyl heptaricacid.

Suitable diamines include but are not limited to those given in theformula below, where R may be an alkyl chain incorporating an variety offunctional groups including ketones, amines, esters, alcohols, ethers,thiols, thioesters, phosphates, phosphonates. The R group is preferablyare alkyl polyamine chain of varying lengths, with examples given inTable 1.

TABLE 1 Diamine: NH₂—R—NH₂; where R may be: —(CH₂)₂—NH—(CH₂)₂—;—(CH₂)₂—NH—(CH₂)₂—NH—(CH₂)₂—; —(CH₂)₂—NH—(CH₂)₂—NH—(CH₂)₂—NH—(CH₂)₂—;—(CH₂)₂—NH—(CH₂)₂—NH—(CH₂)₂—NH—(CH₂)₂—NH(CH₂)₂;

An example of one class of polyamides of the present invention includesthose shown below (x=0-10; n=1-infinity). This also includes allpossible isomers, diastereomers and enantiomers, linear and branchedforms.

Preparation of poly(glycoamidoamine)s

The poly(glycoamidoamine)s are prepared by polycondensation of a diamine[such as diethylenetriamine, triethylenetetramine,tetraethylenepentamine, or pentaethylenehexamine] with a diester ordilactone carbohydrate derivative. After polymerization, each polymerproduct is dissolved in ultra pure water, dialyzed for 24 hours, andlyophilized to dryness. Examples of poly(glycoamidoamine)s which may beprepared by this process are shown below:

Preparation of poly(L-tartaramidoamine)s

Each polymer is synthesized through condensation polymerization of anamine comonomer (AA) with dimethyl L-tartarate (BB) in methanol at roomtemperature to yield a series of AABBAABB copolymers. All productsobtained are of a white color.

Poly(L-tartaramidodiethyleneamine) (T1, see below): Diethylenetriamine(0.29 g, 2.80 mmol) is added to a methanol solution (2.80 mL) ofdimethyl L-tartarate (0.50 g, 2.80 mmol) and stirred for 24 hours atroom temperature. The mixture is dialyzed against ultra pure water toremove monomer, oligomer, and solvent impurities. Yield: 0.38 g, 1.75mmol (62.5%). ¹H NMR (D₂O): δ 4.46 (s, 2H), 3.31 (broad, 4H), 2.69(broad, 4H).

Poly(L-tartaramidotriethylenediamine)(T2, see below):Triethylenetetramine hydrate (containing 20.42% H₂O, 0.41 g, 2.81 mmol)is added and stirred in a methanol solution (2.80 mL) containingdimethyl L-tartarate (0.50 g, 2.80 mmol). After 24 hours, the mixture isdialyzed against ultra pure water. Yield: 0.60 g, 2.31 mmol (82.5%). ¹HNMR (D₂O): δ 4.46 (s, 2H), 3.30 (broad, 4H), 2.68 (broad, 8H).

Poly(L-tartaramidotetraethylenetriamine) (T3, see below): DimethylL-tartarate (0.50 g, 2.80 mmol) is added to a methanol solution (10.0mL) containing triethylamine (1.4 g, 14 mmol) and tetraethylenepentaminepentahydrochloride (1.04 g, 2.80 mmol). After 80 hours, the mixture isdialyzed against ultra pure water. Yield: 0.72 g, 2.38 mmol (84.8%). ¹HNMR (D₂O): δ 4.45 (s, 2H), 3.31 (broad, 4H), 2.70 (broad, 12H).

Poly(L-tartaramidopentaethylenetetramine) (T4, see below): DimethylL-tartarate (0.50 g, 2.80 mmol) is added to a methanol solution (10.0mL) of triethylamine (1.7 g, 17 mmol) and pentaethylenehexaminehexahydrochloride (1.26 g, 2.80 mmol). After 8 hours, the mixture isdialyzed against ultra pure water. Yield: 0.50 g, 1.44 mmol (51.4%). ¹HNMR (D₂O): δ 4.45 (s, 2H), 3.31 (broad, 4H), 2.71 (broad, 16H).

Examples of the poly(L-tartaramidoamine)s are shown below:

Preparation of poly(D-glucaramidoamine)s

Polymerization. Each polymer is synthesized through condensationpolymerization of an amine comonomer with esterified D-glucaric acid inmethanol at room temperature. All products obtained are of a whitecolor.

Poly(D-glucaramidodiethyleneamine) (1, see below): Diethylenetriamine(0.09 g, 0.87 mmol) is added to a methanol solution (8.40 mL) ofesterified D-glucaric acid (0.20 g, 0.84 mmol). The clear solutionbecomes cloudy after approximately 10-15 min while stirring at roomtemperature. After 48 hours, the polymerization mixture is dissolved inand dialyzed against ultra pure water to purity. Yield: 0.22 g, 0.79mmol, 91.2%. ¹H NMR (DMSO-d₆): δ 7.86 (s, 1H), 7.69 (s, 1H), 4.02 (d,1H), 3.96 (d, 1H), 3.88 (t, 1H), 3.72 (t, 1H), 3.17 (s, 4H), 2.58 (s,4H).

Poly(D-glucaramidotriethylenediamine) (2, see below):Triethylenetetramine hydrate (containing 20.42% H₂O, 0.15 g, 0.82 mmol)is added to a methanol solution (8.40 mL) of esterified D-glucaric acid(0.20 g, 0.84 mmol). The clear solution becomes cloudy afterapproximately 10-15 min while stirring at room temperature. After 48hours, the polymerization mixture is dissolved in and dialyzed againstultra pure water to purity. Yield: 0.18 g, 0.56 mmol, 68.6%. ₁H NMR(DMSO-d₆): δ 7.89 (s, 1H), 7.70 (s, 1H), 4.05 (d, 1H), 3.97 (d, 1H),3.89 (t, 1H), 3.73 (t, 1H), 3.20 (s, 4H), 2.60 (s, 8H).

Poly(D-glucaramidotetraethylenetriamine) (3, see below): EsterifiedD-glucaric acid (0.20 g, 0.84 mmol) is added to a methanol solution(8.40 mL) of triethylamine (0.42 g, 4.15 mmol) andtetraethylenepentamine pentahydrochloride (0.31 g, 0.83 mmol). The clearsolution becomes cloudy after approximately 30 min while stirring atroom temperature. After 6 hours, the polymerization mixture is dissolvedin and dialyzed against ultra pure water to purity. Yield: 0.11 g, 0.30mmol, 36.0%. ₁H NMR (DMSO-d₆): δ 7.90 (s, 1H), 7.70 (s, 1H), 4.02 (d,1H), 3.96 (d, 1H), 3.88 (t, 1H), 3.73 (t, 1H), 3.19 (s, 4H), 2.60 (s,12H).

Poly(D-glucaramidopentaethylenetetramine) (4, see below): EsterifiedD-glucaric acid (0.20 g, 0.84 mmol) is added to a methanol solution(5.60 mL) of triethylamine (0.51 g, 5.04 mmol) and pentaethylenehexaminehexahydrochloride (0.38 g, 0.84 mmol). The clear solution becomes cloudyafter approximately 30 min while stirring at room temperature. After 8hours, the polymerization mixture is dissolved in and dialyzed againstultra pure water to purity. Yield: 0.15 g, 0.37 mmol, 44.0%. ₁H NMR(DMSO-d₆): δ 7.87 (s, 1H), 7.72 (s, 1H), 4.02 (d, 1H), 3.94 (d, 1H),3.87 (t, 1H), 3.71 (t, 1H), 3.16 (s, 4H), 2.59 (s, 14H), 2.22 (s, 2H).

Examples of poly(D-glucaramidoamine)s are shown below:

Related to the poly(glycoamidoamine)s, poly(D-glucaramidoamine)s andpoly(L-tartaramidoamine)s are polyamides which include bothanhydride-terminated and carboxylic acid-terminated and mixturesthereof. Examples of a carboxylic acid-terminated polyamides of thepresent invention includes the following (this also includes allpossible isomers, diastereomers and enantiomers), wherein X=0-10, andn=1-infinity).

2) Polymeric Dendritic Polymers

Another class of polymers comprising an oligoamine shell and acyclodextrin core is created by reacting an appropriately substitutedcyclodextrin with a polyamine, such those give in Table 1, to arrive ata cyclodextrin-polyamide structure. Suitable cyclodextrins includealpha, beta and gamma cyclodextrins. These cyclodextrins may besubstituted in such a way as to produce linear and branched polyamidesby reaction with the appropriate polyamine. In such systems, adedrimer-type array would be formed, in which the amine chains would beattached to the cyclodextrin base.

One specific type of dendrimer is a cyclodextrin/oligoamine which hasbeen synthesized by selectively halogenating all of the primary hydroxylgroups around the cyclodextrin ring. An example of an oligoamine-dendronwhich has been prepared by this process and contains a thioacetyl groupis shown below:

Examples of β-cyclodextrin/thiol-diethylentriamine dendrimers preparedby this process include those shown below:

Another example of a dendritic cyclodextrin is a cyclodextrin/triazolesystem (see structures 14 a and 14 b.1-14.b.4). Such dendrimers may beprepared by a copper-catalyzed 1,3-dipolar addition as shown below. Theability of such dendrimers to bind DNA at low N/P ratios suggests a useas a DNA delivery vehicle. Examples of dendrimers prepared by thisprocess include, but are not limited to, the following examples.

One dendrimer is synthesized by 1,3 dipolar addition of acetylatedper-azido beta-cyclodextrin and an alkyne dendron with a copper(I)catalyst. The dendron was synthesized by coupling t-butylcarbonate (BOC)protected ethylenediamine with propiolic acid. All of the products werepurified using silica gel column chromatography. The characterization ofthe precursors and dendron was completed using ¹H NMR, IR, and massspectrometry. The final step in the synthesis of the triazole dendrimerrequired the ‘click reaction’. The azide group present in core moietyundergoes 1,3 dipolar addition with the acetylene group present in thedendron using a copper(I) catalyst. The 1,4 isomer of the triazole isobtained exclusively with this reaction. Finally, through deprotectionof the BOC and purification via column chromatography, pure dendrimer orlinear polymer is obtained. The scheme for the synthesis of the triazoledendrimers 14 is shown in below:

3) 1,3-Dipolar Addition Polymers

A third class of materials are prepared by combining a diazide monomer(usually a carbohydrate diazide) with an a dialkyne unit containingoligamines. This forms a series of polymers that contain triazole unitsbetween the combined monomers along the polymer chain (see below).

4) Carbohydrate-Containing Biodegradable Polyesters:

The fourth class of polymers are biodegradable polyesters. Thesematerials are formed by combining a carbohydrate alkene molecule with aseries of oligoamines. Through protecting the oligoamines, linearpolyesters can be formed. If the oligoamines are not protected, a seriesof hyperbranched polymers can be formed. In addition, most anycarbohydrate molecule may be utilized as the starting material for thisclass of polymers, including monosaccharides and disaccharides andpolysaccharides. With this property in mind, a biodegradable genedelivery system based on an oligoamine and a carbohydrate moiety isprepared, which incorporates an ester bond between repeatedcarbohydrates such as glucose molecules and oligoamine residues. Forthis reaction, any type of carbohydrate, substituted carbohydrate, oroligoamine or substituted oligoamine residues can be used to form anymolecular weight (degree of polymerization) of polymers.

As shown in the reaction scheme above, a diacrylate glucose monomer andBOC (t-butylcarbonate)-protected diethylenetriamine are polymerized inTHF for 48. hours. The polymer is isolated via precipitation with hexaneand dried under the vacuum. Finally, the BOC groups are removed underacidic conditions to yield the deprotected polymer structure Thedegradation time can vary significantly according to the degree ofhydrophobicity of the polyester structure; hydrophobic polymers willdecrease the rate of hydrolysis.

Polyplex Formation

Generally, the polyplexes are formed by mixing a biologically activemolecule where preferably the biologically active molecule is anoligonucleotide or polypeptide, for instance, pDNA (plasmid DNA), withan appropriate volume of polymer dissolved in nuclease-free water toyield a final N/P ratio of about 20 to about 60, or from about 25 toabout 35, or alternatively, an N/P ratio of about 30. The polyplex sizefor the majority of the polyplexes is between about 50 to about 650 nm.In general, as the number of amine units between each polymer increases,the binding affinity increases and the size of the polyplexes produceddecreases.

The amine number and the hydroxyl stereochemistry mediate the compactionof pDNA into nanoparticles. In general, as the number of amine unitsbetween each polymer increases, the polymer-pDNA binding affinityincreases and the size of the polyplexes produced decreases. In general,the galactarate polymers are the most efficient and the D-mannaratepolymers are the least efficient to compact pDNA.

The optimum concentration of polyplex formulation to promote thesmallest polyplex formation is at a final concentration of about 0.01mg/mg of nucleic acid (after it is mixed with each polymer at a chargeratio of 10+/−), all of the polymers formed polyplexes in the properrange to be endocytosed. The polymers compact pDNA into polyplexesbetween about 75 to about 170 nm. The N/P ratios used for this arechosen based on a common N/P ratio in which all of the polymersinhibited DNA migration on the gel shift assay for each polymer. Eachsample is analyzed by dynamic light scattering using a BrookhavenZetaPals Instrument (Holtzville, N.Y.). The particle size of thepolyplex is determined as a function of pDNA concentration. The polymersdiscussed above are able to compact DNA into polyplexes small enough tobe endocytosed in the cell culture studies described below.

Polyamides For Nucleic Acid Delivery

In the description that follows, a number of terms used in molecularbiology and medical/pharmaceutical sciences are utilized extensively. Inorder to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided. Under these definitions, thefollowing terms have the following meaning unless otherwise specifiedherein:

Association: the covalent or non-covalent joining of two or moremolecules, which may occur permanently, temporary, or transiently. Amolecular complex is formed by the stable or semi-stable association oftwo or more compounds.

Base Pair (bp): a partnership of adenine (A) with thymine (T), or ofcytosine (C) with guanine (G) in a double stranded DNA molecule. In RNA,uracil (U) is substituted for thymine. Base pairs are the to be“complementary” when their component bases pair up normally when a DNAor RNA molecule adopts a double stranded configuration.

The term “biologically active molecule” as used herein, refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive siNA molecules either alone or in combination with othermolecules contemplated by the instant invention include therapeuticallyactive molecules such as antibodies, cholesterol, hormones, antivirals,peptides, proteins, chemotherapeutics, small molecules, vitamins,co-factors, nucleosides, nucleotides, oligonucleotides, enzymaticnucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example, lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

Cellular Delivery (also referred to herein interchangeably andequivalently as “delivery”): a process by which a desired compound istransferred to a target cell such that the desired compound isultimately located inside the target cell, or in or on the target cellmembrane. In certain uses delivery to a specific target cell type ispreferable.

Cellular Delivery Molecule: a molecule that mediates the CellularDelivery of itself, a molecular complex comprising the Cellular DeliveryMolecule, and/or a molecule comprising the Cellular Delivery Molecule.

Cell delivery polymer: a polymer that functions as a Cellular DeliveryMolecule, either by itself, as a part of a molecular complex. By way ofnon-limiting example, Cell delivery polymers include polyamides,dendritic macromolecules, and biodegradable polyesters.

Complementary Nucleotide Sequence: a sequence of nucleotides in asingle-stranded molecule of DNA or RNA that is sufficientlycomplementary to another single strand to specifically (non-randomly)hybridize to it with consequent hydrogen bonding.

Construct: a vector sequence, or a portion thereof, that has been linkedwith one or more non-vector sequences.

Inducer: a molecule that triggers gene transcription by binding to aregulator protein such as a repressor. Induction: the switching on oftranscription as a result of interaction of an inducer with a positiveor negative regulator.

Negative Regulation of Transcription: a mechanism of control of geneexpression where a gene is transcribed unless transcription is preventedby the action of a negative regulator, or repressor.

Nucleotide: a base-sugar-phosphate combination. Nucleotides aremonomeric units of a nucleic acid sequence (DNA and RNA). Nucleotidesmay also include mono-, di- and triphosphate forms of such nucleotides.The term nucleotide includes ribonucleoside triphosphates ATP, UTP, T,CTG, GTP and deoxyribonucleoside triphosphates such as DATP, dCTP, dITP,dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, forexample, [aS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotidederivatives that confer nuclease resistance on the nucleic acid moleculecontaining them. The term nucleotide as used herein also refers todideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.Illustrated examples of dideoxyribonucleoside triphosphates include, butare not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. According tothe present invention, a “nucleotide” may be unlabeled or detectablylabeled by well known techniques. Detectable labels include, forexample, radioactive isotopes, fluorescent labels, chemiluminescentlabels, bioluminescent labels and enzyme labels. Various labelingmethods known in the art can be employed in the practice of thisinvention.

Nucleotide Analog: a purine or pyrimidine nucleotide that differsstructurally from an A, T, G, C, or U base, but is sufficiently similarto substitute for the normal nucleotide in a nucleic acid molecule.Inosine (I) is a nucleotide analog that can hydrogen bond with any ofthe other nucleotides, A, T, G, C, or U. In addition, methylated basesare known that can participate in nucleic acid hybridization. Methods ofpreparing and using modified oligonucleotides are described in: Verma S,Eckstein F. Modified oligonucleotides: synthesis and strategy for users.Annu Rev Biochem. 1998; 67:99-134. By way of non-limiting example,nucleotide analogs include 2,6-diamino purine, 6-methyladenine,8-azaguanine, 5-bromouracil, 5-hydroxymethyl uracil, 5-methylcytosine(5MC), 5-hydroxymethylcytosine (HMC), 8-chloroadenosine, glycosyl HMC,and gentobiosyl HMC. Fluorescent nucleotide analogs, such as thosedescribed by Jameson and Eccleston (Fluorescent nucleotide analogs:synthesis and applications. Methods Enzymol. 1997; 278:363-90), andcyclic nucleotide analogs, such as those described by Schwede et al.(Cyclic nucleotide analogs as biochemical tools and prospective drugs.Pharmacol Ther 2000 87(2-3):199-226) may also be used in the invention.

Nucleic Acid: As used herein “nucleic acid” and its grammaticalequivalents will include the full range of polymers of single or doublestranded nucleotides. A nucleic acid typically refers to apolynucleotide molecule comprised of a linear strand of two or morenucleotides (deoxyribonucleotides and/or ribonucleotides) or variants,derivatives and/or analogs thereof. The exact size will depend on manyfactors, which in turn depends on the ultimate conditions of use, as iswell known in the art. The nucleic acids of the present inventioninclude without limitation primers, probes, oligonucleotides, vectors,constructs, plasmids, genes, transgenes, genomic DNA, cDNA, PCRproducts, restriction fragments, and the like.

Promoter: As used herein, a promoter is an example of a transcriptionalregulatory sequence, and specifically is a DNA sequence generallydescribed as the 5′-region of a gene located proximal to the startcodon. The transcription of an adjacent DNA segment is initiated at thepromoter region. A repressible promoter's rate of transcriptiondecreases in response to a repressing agent. An inducible promoter'srate of transcription increases in response to an inducing agent. Aconstitutive promoter's rate of transcription is not specificallyregulated, though it can vary under the influence of general metabolicconditions.

Recognition sequence: As used herein, a recognition sequence is aparticular sequence to which a protein, chemical compound, DNA, or RNAmolecule (e.g., restriction endonuclease, a modification methylase, or arecombinase) recognizes and binds. In the present invention, arecognition sequence will typically, but need not, refer to arecombination site. For example, the recognition sequence for Crerecombinase is loxP which is a 34 base pair sequence comprised of two 13base pair inverted repeats (serving as the recombinase binding sites)flanking an 8 base pair core sequence. See FIG. 1 of Sauer, B., CurrentOpinion in Biotechnology 5:521-527 (1994). Other examples of recognitionsequences are the attB, attP, attL, and attR sequences which arerecognized by the recombinase enzyme Integrase. The attB site is anapproximately 25 base pair sequence containing two 9 base pair core-typeInt binding sites and a 7 base pair overlap region. The attP site is anapproximately 240 base pair sequence containing core-type Int bindingsites and arm-type Int binding sites as well as sites for the auxiliaryproteins integration host factor (IHF), FIS and excisionase (Xis). SeeLandy, Current Opinion in Biotechnology 3:699-707 (1993). Such sites mayalso be engineered according to the present invention to enhanceproduction of products in the methods of the invention. When suchengineered sites lack the P1 or H1 domains to make the recombinationreactions irreversible (e.g., attR or attP), such sites may bedesignated attR′ or attP′ to indicate that the domains of these siteshave been modified in some way.

Repressor: a protein which prevents transcription by binding to aspecific site on DNA.

Target Cell: any cell to which a desired compound is delivered. Cells towhich the delivery methods of this invention can be applied includecells in vitro, cells ex vivo or cells in vivo. Target cells may be incell culture, on tissue culture, in any form of immobilized state, orgrown on liquid, semi-solid or solid medium. Target cells may be in theform of a monolayer. Target cells may be collected from an organismand/or cultured by any known method. Target cells include cells withoutcell walls and cells from which cell walls have been removed by anyknown treatment (e.g., formation of protoplasts) from which viable cellscan be recovered.

Transcriptional regulatory sequence: As used herein, transcriptionalregulatory sequence is a functional stretch of nucleotides contained ona nucleic acid molecule, in any configuration or geometry, that acts toregulate the transcription of one or more structural genes intomessenger RNA. Examples of transcriptional regulatory sequences include,but are not limited to, promoters, enhancers, repressors, and the like.“Transcription regulatory sequence,” “transcription sites” and“transcription signals” may be used interchangeably.

Transfection: the delivery of expressible nucleic acid to a target cell,such that the target cell is rendered capable of expressing the nucleicacid. It will be understood that the term “nucleic acid” includes bothDNA and RNA without regard to molecular weight, and the term“expression” means any manifestation of the functional presence of thenucleic acid within the cell including, without limitation, bothtransient expression and stable expression.

Transfection Agent: any substance which provides significant enhancementof transfection (2-fold or more) over transfection compositions that donot comprise the transfection agent.

Vector: As used herein, a vector is a nucleic acid molecule thatprovides a useful biological or biochemical property to a nucleic acidsequence or molecule of interest, for example, an Insert, a codingregion, etc. Examples include plasmids, phages, autonomously replicatingsequences (ARS), centromeres, and other nucleic acid sequences that areable to replicate or be replicated in vitro or in a host cell, or toconvey a desired nucleic acid segment to a desired location within ahost cell. A vector may comprise various structural and/or functionalsequences, for example, one or more restriction endonuclease recognitionsites at which the vector sequences can be manipulated in a determinablefashion without loss of an essential biological function of the vector,and into which a nucleic acid fragment can be inserted, for example tobring about its replication and/or cloning. Vectors can further provideprimer sites, e.g., for PCR, transcriptional and/or translationalinitiation and/or regulation sites, recombinational signals, replicons,selectable markers, and other sequences known to those skilled in theart. A vector comprising a nucleic acid insert is a Construct. Thus, agene therapy construct is a gene therapy vector into which a therapeuticgene has been cloned. Similarly, a construct that expresses an antisensetranscript is an “antisense construct.”

Biologically Active: As used herein, the term “biologically active”(synonymous with “bioactive”) indicates that a composition or compounditself has a biological effect, or that it modifies, causes, promotes,enhances, blocks, or reduces a biological effect, or which limits theproduction or activity of, reacts with and/or binds to a second moleculethat has a biological effect. The second molecule can, but need not, beendogenous. A “biological effect” may be but is not limited to one thatstimulates or causes an immunoreactive response; one that impacts abiological process in a cell, tissue or organism (e.g., in an animal);one that impacts a biological process in a pathogen or parasite; onethat generates or causes to be generated a detectable signal; and thelike. Biologically active compositions, complexes or compounds may beused in investigative, therapeutic, prophylactic and diagnostic methodsand compositions. Biologically active compositions, complexes orcompounds act to cause or stimulate a desired effect upon a cell,tissue, organ or organism (e.g., an animal). Non-limiting examples ofdesired effects include modulating, inhibiting or enhancing geneexpression in a cell, tissue, organ, or organism; preventing, treatingor curing a disease or condition in an animal suffering therefrom;limiting the growth of or killing a pathogen in an animal infectedthereby; augmenting the phenotype or genotype of an animal; stimulatinga prophylactic immunoreactive response in an animal; or diagnosing adisease or disorder in an animal.

In the context of investigative applications of the invention, includingbut not limited to forensic and scientific research applications, theterm “biologically active” indicates that the composition, complex orcompound has an activity that results, directly or indirectly, in achange in some form of measurable output in materials, biologicalsamples, cells or organisms that have been contacted therewith.Investigative applications may be used to determine the quantity orconcentration of a selected target compound in a test sample, todetermine the effect of a bioactive compound upon cells or animals, orto screen for compounds having an activity that alters, blocks oraugments a selected biological activity.

In the context of therapeutic applications of the invention, the term“biologically active” indicates that the composition, complex orcompound has an activity that impacts an animal suffering from a diseaseor disorder in a positive sense and/or impacts a pathogen or parasite ina negative sense. Thus, a biologically active composition, complex orcompound may cause or promote a biological or biochemical activitywithin an animal that is detrimental to the growth and/or maintenance ofa pathogen or parasites; or of cells, tissues or organs of an animalthat have abnormal growth or biochemical characteristics, such as cancercells.

In the context of prophylactic applications of the invention, the term“biologically active” indicates that the composition or compound inducesor stimulates an immunoreactive response. In some preferred embodiments,the immunoreactive response is designed to be prophylactic, i.e., toprevent infection by a pathogen. In other preferred embodiments, theimmunoreactive response is designed to cause the immune system of ananimal to react to the detriment of cells of an animal, such as cancercells, that have abnormal growth or biochemical characteristics. In thisapplication of the invention, compositions, complexes or compoundscomprising antigens are formulated as a vaccine.

In the context of diagnostic applications on the invention, the term“biologically active” indicates that the composition, complex orcompound can be used for in vivo or ex vivo diagnostic methods and indiagnostic compositions and kits. For diagnostic purposes, a preferredbiologically active composition or compound is one that can be detected,typically (but not necessarily) by virtue of comprising a detectablepolypeptide. Antibodies to an epitope found on composition or compoundmay also be used for its detection. It will be understood by thoseskilled in the art that a given composition, complex or compound may bebiologically active in therapeutic, diagnostic and/or prophylacticapplications. A composition, complex or compound that is described asbeing “biologically active in a cell” is one that has biologicalactivity in vitro (i.e., in a cell or tissue culture) or in vivo (i.e.,in the cells of an animal). A “biologically active component” of acomposition or compound is a portion thereof that is biologically activeonce is liberated from the composition or compound. It should be notedthat such a component may also be biologically active as a moiety orother portion of the composition or compound.

In the disclosure and the claims, “and/or” means additionally oralternatively. Moreover, any use of a term in the singular alsoencompasses plural forms.

Other terms used in the fields of recombinant DNA technology, molecularand cell biology, and the medical/pharmaceutical arts, as used herein,are intended to encompass the broadest scope term understood in the artfor a given and will be generally understood by one of ordinary skill inthe applicable arts.

In one embodiment, the invention encompasses a method of delivering abiologically active molecule to a cell, comprising contacting the cellwith (a) a biologically active molecule and (b) a cellular deliverypolymer.

In one embodiment, present invention also provides for compositions andnon-covalent complexes comprising one or more polymers of the presentinvention, e.g., polyamides, dendritic macromolecules (polymerscomprising an oligoamine shell and a cyclodextrin core), andcarbohydrate-containing degradable polyesters, and at least one nucleicacid molecule (e.g., one or more oligonucleotides) or at least onepolypeptide or both. The invention also provides compositions comprisingsuch complexes.

Complexes according to the invention or portions thereof, can comprise acellular delivery molecule or agent that can facilitate thetranslocation of the complex or portion thereof into cells. In someembodiments, cellular delivery molecules for use in the presentinvention may comprise one or more one or more polymers of the presentinvention, e.g., polyamides, dendritic macromolecules (polymerscomprising an oligoamine shell and a cyclodextrin core), andcarbohydrate-containing degradable polyesters.

In some embodiments, a cell, tissue, organ or organism may be contactedwith a complex of the invention. Preferably, the complex is taken up bythe cell or by one or more cells of the tissue, organ or organism.

In another exemplary and non-limiting embodiment of the invention,compositions comprising complexes between cellular delivery polymers andoligonucleotides are formed and can be applied to cultured mammaliancells. The complex may also comprise a combination of labeled andnonlabeled nucleic acid and or peptide. These complexes allow mediationof an activity associated with the oligonucleotide, which, by way ofnon-limiting example, can be a gene-containing oligonucleotide, anantisense oligonucleotide, an aptamer, a short interfering RNA (siRNA),a short hairpin RNA (shRNA), a small temporally regulated RNA (stRNA),and the like. In some embodiments, oligonucleotides are preferred.

In other specific embodiments, the biologically active molecule and/orcell delivery agent is covalently labeled with a fluorophores(fluorescent moiety), for example with fluorescein or a derivative offluorescein.

In another embodiment, the compositions may comprise one or morefluorescent molecules or moieties, which may be the same or different,and may be covalently attached to one or more polypeptides and/ornucleic acid molecules in the complexes of the invention. Alternatively,or in addition, complexes of the invention may comprise one or more“free” fluorescent molecule (i.e., one or more fluorescent moleculesthat are not covalently attached to either the polypeptide or theoligonucleotide but may still be associated with the complex). One ormore of the compounds of the compositions or complexes can be abiologically active molecule.

Kits according to the invention may further comprise one or moretransfection agents, one or more cells, one or more nucleic acids, oneor more set of instructions, and one or more biologically activemolecules.

Other additional kit components include without limitation: additionalnucleic acids, such as oligonucleotides, iRNA molecules, plasmids, etc.;one or more recombinases, including without limitation site-specificrecombinases; one or more recombination proteins; and/or one or morecells. In some embodiments, the cells are competent for transfection ortransformation.

In other embodiments, the invention provides a complex comprising a celldelivery polymer and a biologically active agent that is desirably takenup by cells, wherein the cell delivery polymer or biologically activeagent comprises a fluorescent moiety.

The nucleic acid of the complexes and other embodiments of the inventioncan comprise from 5 bases to about 200 kilobases. Any type of nucleicacid may be used, including by way of non-limiting example mRNA, tmRNA,tRNA, rRNA, siRNA, shRNA, PNA, ssRNA, dsRNA, ssDNA, dsDNA, DNA:RNAhybrid molecules, plasmids, artificial chromosomes, gene therapyconstructs, cDNA, PCR products, restriction fragments, ribozymes,antisense constructs, and combinations thereof. Reviews of tmRNA includeMuto A, Ushida C, Himeno H. A bacterial RNA that functions as both atRNA and an mRNA. Trends Biochem Sci. 1998 January; 23(1):25-9; andWithey J H, Friedman D I. The biological roles of trans-translation.Curr Opin Microbiol. 2002 April; 5(2): 154-9). The nucleic acid maycomprise one or more chemical modifications.

A complex according to the invention may further comprise one or moretransfection agents, one or more recombinases and, additionally oralternatively, one or more recombination proteins.

A nucleic acid used in the invention includes, in some embodiments, asequence that encodes a protein or a portion thereof. In someembodiments, a cellular nucleic acid encoding the protein, or a portionthereof, is desirably replaced by the sequence in one form of genetherapy. Additionally or alternatively, the protein is expressed in thecell. The protein may be exogenous or endogenous. In the latter case,the cells to be transfected may comprise a non-functional form of theprotein.

A composition of the invention may be a pharmaceutical composition. Incertain embodiments, the biologically active molecule is one or more ofthe nucleic acids that has a biological activity, including but notlimited to therapeutic activity. By way of non-limiting example,biologically active nucleic acids are selected from the group consistingof mRNA, tmRNA, tRNA, rRNA, siRNA, shRNA, PNA, ssRNA, dsRNA, ssDNA,dsDNA, DNA:RNA hybrid molecules, plasmids, artificial chromosomes, genetherapy constructs, cDNA, PCR products, restriction fragments,ribozymes, antisense constructs, and combinations thereof.

Additionally or alternatively, polypeptide of the complex isbiologically active. A biologically active polypeptide may be atherapeutic protein. By way of non-limiting example, bioactive proteinsinclude antibodies or antibody fragments, hormones, enzymes,transcription factors, growth factors, and the like.

The invention further provides a method of providing gene therapy to anindividual in need thereof, of treating an individual suffering from adisease or disorder, the method comprising contacting the individual, orcells therefrom, with one or more complexes, compositions and/orpharmaceutical compositions of the invention.

The invention further provides a method of testing a cellular responseto a test compound, the method comprising: (a) contacting a first cellwith, in any order or combination, a biologically active molecule and acellular delivery polymer, (b) contacting a second cell with, in anyorder or combination, a second biologically active molecule and thecellular delivery polymer, (c) contacting the cells with the testcompound, before (a); during (a) or (b); between (a) and (b); and,additionally or alternatively, after (b); (d) measuring and comparing atleast one parameter of from the first cell with the signal from thesecond cell. In certain embodiments, one or more of the cells compriseone or more reporter genes that generate a detectable signal orinterfere with the production of a detectable signal.

In one embodiment, the present invention provides for a new series ofpolyamides for use as gene delivery agents. These polymers bindproducts, e.g., oligonucliotides, and facilitate cellular uptake. In oneembodiment, the invention provides for the in vitro delivery of plasmidDNA into cells. In one embodiment, the invention provides for the invivo delivery of plasmid DNA into cells.

In one embodiment, the present invention relates to the use ofpolyamides for delivering nucleic acids into a cell. In one embodiment,the nucleic acid is an oligonucleotide. In another embodiment, theoligonucleotide contains from about 10 to about 1000 nucleotides. Inanother embodiment, the oligonucleotide is an antisense oligonucleotideor oligodeoxynucleotide. In another embodiment, the oligonucleotideis anoligonucleotide, an antisense oligonucleotide residue oroligodeoxynucleotide residue.

In another embodiment, the nucleic acid is selected from the groupconsisting of antisense constructs, antisense polynucleotide, artificialchromosomes, cDNA, concatemers, concatemeric decoy oligonucleotides, CpGoligomers, cyclic oligonucleotides, decoy oligonucleotides, DNA:RNAhybrid molecules, dsDNA, dsRNA, gene therapy constructs, LNA,morpholinos, mRNA, oligonucleotides and oligodeoxynucleotides withphosphorodiester backbones or phosphorothioate backbones, PCR products,plasmids, PNA, restriction fragments, ribozyme, RNA, RNAi, RNAi inducingpolynucleotide, rRNA, shRNA, siRNA, spiegelmers, ssDNA, ssRNA, tmRNA,transgenes, tricyclo-DNA, triple helices, tRNA, and combinationsthereof.

In one specific embodiment, the present invention provides polymercompositions, complexes and methods for delivering one or more nucleicacids (e.g., one or more nucleic acid molecules, oligonucleotides,polynucleotides, vectors, genes and the like) and/or one or morepeptides (e.g., one or more peptides, oligopeptides, polypeptides,proteins or protein complexes) to cells, tissues, organs and wholeorganisms. The compositions and complexes of the invention typicallycomprise one or more nucleic acids and/or one or more proteins orpolypeptides (which can be cellular delivery (suitably, translocating)peptides, polypeptides or proteins.

In certain such aspects of the invention, the complexes comprising oneor more nucleic acids and/or one or more peptides are delivered to andtaken up by the cells, tissues, organs or organisms, and cells, tissues,organs or organisms. The invention also provides compositions comprisingthe polymer complexes of the invention and one or more additionalcomponents. Suitable such compositions, for example, includepharmaceutical compositions comprising one or more of the complexes ofthe invention and one or more pharmaceutically acceptable carriers,excipients or diluents therefor. The invention also provides methods forproducing such complexes and compositions, and methods of using suchcomplexes and compositions to deliver one or more nucleic acid moleculesand/or one or more peptides to cells, tissues, organs or organisms, forexample for therapeutic or prophylactic purposes. The invention alsoprovides kits comprising the complexes and compositions of theinvention, and optionally further comprising one or more additionalcomponents suitable for use in or with the complexes and compositions,and/or for carrying out the methods, of the present invention.

In one embodiment, the present invention relates to the use of nucleicacids. In one embodiment, the nucleic acid is an oligonucleotide. Inanother embodiment, the oligonucleotide contains from about 10 to about1000 nucleotides. In another embodiment, the oligonucleotide is anantisense oligonucleotide or oligodeoxynucleotide. In anotherembodiment, the oligonucleotideis an oligonucleotide, an antisenseoligonucleotide residue or oligodeoxynucleotide residue.

In another embodiment, the nucleic acid is selected from the groupconsisting of antisense constructs, antisense polynucleotide, artificialchromosomes, cDNA, concatemers, concatemeric decoy oligonucleotides, CpGoligomers, cyclic oligonucleotides, decoy oligonucleotides, DNA:RNAhybrid molecules, dsDNA, dsRNA, gene therapy constructs, LNA,morpholinos, mRNA, oligonucleotides and oligodeoxynucleotides withphosphorodiester backbones or phosphorothioate backbones, PCR products,plasmids, PNA, restriction fragments, ribozyme, RNA, RNAi, RNAi inducingpolynucleotide, rRNA, shRNA, siRNA, spiegelmers, ssDNA, ssRNA, tmRNA,transgenes, tricyclo-DNA, triple helices, tRNA, and combinationsthereof.

The present invention provides a new class of non-viral transductionvectors that can be used for both in vivo and in vitro applications. Inparticular, these vectors can be used for gene transfer applications.These new gene transduction vectors can achieve transfer efficienciesfar greater to commercially available polymeric and liposomal genetransfer vectors while maintaining little or no toxicity in vitro. Theirlow in vitro toxicity makes them ideal candidates for in vivo use. Thepresent invention provides a gene transfer vector that has comparableefficiency to a viral vector without the potential for alife-threatening immune response.

Furthermore, the unique polycationic structure of these polymersassociates with many suitable bioactive molecules, including proteinsand other compounds that poses multiple cationic sites. The polymer canact as a delivery vehicle for the associated bioactive molecule, in vivoor in vitro, to the cells of interest for the bioactive molecule.

In one embodiment, the present invention provides for a new series ofpolyamides for use as gene delivery agents. These polymers bindproducts, e.g., oligonucliotides, and facilitate cellular uptake. In oneembodiment, the invention provides for the in vitro delivery of plasmidDNA into cells.

Polypeptides

As noted above, the compositions and complexes of the present inventioncomprise one or more peptides, polypeptides or proteins. In certainaspects of the invention, the peptides, polypeptides or proteins used inthese complexes and compositions are peptides, polypeptides or proteinsthat are to be delivered to cells, tissues, organs or organisms for anysuitable biological, therapeutic and/or prophylactic purpose.

As used herein, the term “polypeptide” includes without limitationpeptides (oligopeptides), proteins, and polypeptides. All of these arepolymers of two or more amino acids joined by an amino bond. Generally,peptides comprise from 2 to about a amino acid residues, wherein “a” isany whole integer between 5 and 50, preferably between 10 and 30, andmay be isolated from natural sources or more typically are synthesizedin vitro. As used herein, the term “oligopeptide” may be usedinterchangeably and equivalently with the term “peptide” as definedabove. As used herein, “polypeptides” generally comprise about b aminoacids, wherein “b” is any whole integer between 25 and 50,000,preferably between 50 and 10,000, and more preferably between 50 and1,000. The term “protein” encompasses polypeptides, as well as complexesof two or more covalently or non-covalently bonded polypeptides.Polypeptides and proteins are purified from their natural sources and/orare synthesized using recombinant DNA technology.

Peptides, polypeptides, proteins and protein complexes suitable for usein the complexes, compositions and methods of the present inventioninclude any peptide, polypeptide, protein and protein complex, orportion thereof, that has a desired biological or physiological effecton the cells, tissues, organs and organisms to which the peptides,polypeptides, proteins and protein complexes are delivered. Non-limitingexamples of such peptides, polypeptides, proteins and protein complexesinclude: enzymes, e.g., Idnases; peptidases/proteinases;oxidoreductases; nucleases; recombinases (including Cre, Int, Flp, Tn5resolvase, and the like); ligases (including DNA ligases and the like);lyases; isomerases (including topoisomerases and the like); polymerases(including DNA polymerases, RNA polymerases, reverse transcriptases, andthe like); transferases (including terminal transferases, glutathioneS-transferases, and the like); ATPases; GTPases; etc.; cytokines, e.g.,growth factors (such as epidermal growth factor (EGF), fibroblast growthfactors (FGFs), keratinocyte growth factors (KGFs), hepatocyte growthfactors (HGFs), platelet-derived growth factor (PDGF), transforminggrowth factors alpha and beta (TGF-α and TGF-β), neurotrophic factor(NTF), ciliary neurotrophic factor (CNTF), brain-derived neurotrophicfactor (BDNTF), glial-derived neurotrophic factor (GDNTF), bonemorphogenic proteins (BMPs), and the like, and variants thereof);interleukins (such as IL-1 through IL-18, and the like, and variantsthereof); interferons (such as IFNα IFN-β, IFN-γ, and the like, andvariants thereof); colony-stimulating factors (such as granulocytecolony-stimulating factor (G-CSF), macrophage colony-stimulating factor(M-CSF), granulocyte-macrophase colony-stimulating factor (GM-CSF);erythropoietin (Epo); thrombopoietin (Tpo); leukemia inhibitory factor(LIF/Steel Factor); tumor-necrosis factors (TNFs); and the like, andvariants thereof); peptide hormones (such as antidiuretic hormone,chorionic gonadotropin, leutenizing hormone, follicle-stimulatinghormone, insulin, prolactin, somatomedins, growth hormone,thyroid-stimulating hormone, placental lactogen, and the like, andvariants thereof); etc.; intracellular signalling peptides; receptors(e.g., cytokine receptors, hormone receptors, antibody receptors,integrins and other extracellular matrix receptors, neurotransmitterreceptors, viral receptors, and the like, and variants thereof);antibodies (e.g., polyclonal or monoclonal antibodies, fragments thereof(including Fab and Fc fragments and portions thereof), andmulti-antibody complexes); vaccine components (including, but notlimited to, proteins or peptides of etiologic agents such as viruses,bacteria, fungi (including yeasts), parasites and the like; proteins orpeptides of tumor cells or other cancer-related proteins or peptides;and other proteins or peptides against which it is desirable to producean immune response in an animal, suitably a mammal such as a human);structural and/or functional proteins or peptides (e.g., hemoglobin,albumins including serum albumins, cytoskeletal proteins, transmembranechannel proteins or peptides, and the like, and fragments or variantsthereof); synthetic peptides (e.g. hexahistidine, polylysine, and othersynthetic peptides of any length containing a desired sequence of two ormore amino acids linked together by peptide bonds to form a peptide,oligopeptide, polypeptide or protein, any and all of which can beproduced by art-known methods of synthetic peptide synthesis that willbe familiar to the ordinarily skilled artisan, and that are describedherein); and the like. Of course, other suitable peptides,oligopeptides, polypeptides and proteins suitable for use in accordancewith the present invention (i.e., in the complexes, compositions andmethods of the invention) will be familiar to one of ordinary skill andtherefore are encompassed by the present invention.

Amino Acids

The term “amino acid” as used herein refers generally to a moleculehaving both a carboxyl (—COOH) and an amino (—NH₂) group attached to thesame carbon atom, called the alpha-carbon atom. Amino acids can berepresented by the general formula R—CH(NH₂)COOH, wherein R is a sidechain or residue which may or may not occur naturally. Generally, theside chain (R) of an amino acid contains c carbon atoms, d nitrogenatoms, 0, 1 or 2 sulfur atoms, d oxygens, and/or d halogen atoms,wherein “c” is any whole integer from 0 to about 20, and “d” is anywhole integer from 0 to about 5.

The terms “natural amino acid” and “naturally-occurring amino acid”refer to Ala, Asp, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr. “Unnatural amino acids”(i.e., amino acids do not occur naturally) include, by way ofnon-limiting example, homoserine, homoarginine, citrulline,phenylglycine, taurine, iodotyrosine, seleno-cysteine, norleucine(“Nle”), norvaline (“Nva”), beta-Alanine, L- or D-naphthalanine,ornithine (“Orn”), and the like.

Amino acids also include the D-forms of natural and unnatural aminoacids. “D-” designates an amino acid having the “D” (dextrorotary)configuration, as opposed to the configuration in the naturallyoccurring (“L-”) amino acids. Where no specific configuration isindicated, one skilled in the art would understand the amino acid to bean L-amino acid. The amino acids can, however, also be in racemicmixtures of the D- and L-configuration. Natural and unnatural aminoacids can be purchased commercially (Sigma Chemical Co.; AdvancedChemtech) or synthesized using methods known in the art. Amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as their biological activityis retained.

Peptide Synthesis

Peptides used in accordance with the present invention may be producedby a variety of methods that will be familiar to those of ordinary skillin the art. For reviews and enabling disclosures of peptide synthesis,see M. Bodanzsky, “Principles of Peptide Synthesis,” 1st and 2nd reviseded., Springer-Verlag, New York, N.Y., 1984 and 1993; Stewart and Young,“Solid Phase Peptide Synthesis,” 2nd ed., Pierce Chemical Co., Rockford,Ill., 1984; Fox J E. Multiple peptide synthesis. Mol. Biotechnol.3:249-258, 1995; Kiso Y, Fujii N, Yajima H. New disulfide bond-formingreactions for peptide and protein synthesis. Braz J Med Biol Res.27:2733-2744, 1994; Bongers J, Heimer E P. Recent applications ofenzymatic peptide synthesis. Peptides. 15:183-193, 1994; Wade J D,Tregear G W. Solid phase peptide synthesis: recent advances andapplications. Australas Biotechnol. 3:332-336, 1993; Fields G B, Noble RL. Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonylamino acids. Int J Pept Protein Res. 35:161-214, 1990; Newton R, Fox JE. Automation of peptide synthesis. Adv Biotechnol Processes. 10:1-24,1988; Barany G, Kneib-Cordonier N, Mullen D G. Solid-phase peptidesynthesis: a silver anniversary report. Int J Pept Protein Res.30:705-739, 1987; Bodanszky M. In search of new methods in peptidesynthesis. A review of the last three decades. Int J Pept Protein Res.25:449-474, 1985; Chaiken I M. Semisynthetic peptides and proteins. CRCCrit. Rev Biochem. 11:255-301, 1981; Fridkin M, Patchomik A. Peptidesynthesis. Annu Rev Biochem. 43:419-443, 1974; Merrifield R B.Solid-phase peptide synthesis. Adv Enzymol Relat Areas Mol. Biol.32:221-296, 1969; and U.S. Pat. No. 4,748,002 (Semi-automatic,solid-phase peptide multi-synthesizer and process for the production ofsynthetic peptides by the use of the multi-synthesizer) to Neimark etal.

Fusion Proteins

In certain embodiments, the peptides, polypeptides or proteins used inthe present invention are in the form of fusion proteins. As usedherein, the term “fusion protein” refers to a peptide, polypeptide orprotein comprising a series of contiguous amino acids from one peptide,polypeptide or protein that are linked via peptide bonds to a series ofcontiguous amino acids from one or more additional peptides,polypeptides or proteins. For example, fusion of the glutathioneS-transferase (GST) domain to a peptide, polypeptide or protein ofinterest allows the fusion protein to be purified by affinitychromatography on glutathione agarose (Pharmacia, Inc., 1995 catalog).The fusion protein may include one or more accessory sequences whichfunction for detection, purification or cleavage of the fusion protein.If the peptide, polypeptide or protein of interest is fused to a seriesof consecutive histidines (for example 6xHis), the fusion protein can bepurified by affinity chromatography on chelating resins containing metalions (Qiagen, Inc.). Fusion proteins may include sequences whichfunction as a protein tag, such as an antibody epitope (e.g., derivedfrom Myc), a thiorescent peptide or a poly Histag. Tags and otherelements may function in the purification and/or detection of the fusionprotein. In producing fusion proteins according to this aspect of theinvention, it is often desirable to compare amino terminal and carboxyterminal fusions for activity, solubility, stability, and the like.

Targeting sequences are another type of accessory element that can becomprised in a fusion protein. Cellular targeting elements, which directfusion proteins to specific cell types, include such things as antibodyfragments directed to a cellular surface molecule, fragments of ligandsfor receptors present on a cell, cell-specific targeting sequencesderived from pathogens, derivatives of cellular adhesion molecules, andthe like. Intracellular targeting elements, which direct fusion proteinsto subcellular locations including, without limitation, the nucleus, thecell membrane, the chloroplast, the mitochondrion, the endoplasmicreticulum, the cytoplasm, and membranes or intermembrane spaces of anyof the preceding, are known and are commercially available (e.g.,Invitrogen's line of pShooter vectors). Various targeting sequences areknown in the art and can be readily incorporated into fusion proteinsusing methods known in the art. Polynucleotides encoding fusion proteinsmay be constructed by standard molecular biology techniques (J.Sambrook, E. F. Fritsch and T. Maniatis (1989). Molecular Cloning, ALaboratory Manual. Cold Spring Harbor Laboratory Press. Cold SpringHarbor, N.Y.).

DNA-Binding Peptides and Proteins

A variety of DNA-binding proteins, particularly those that are basic,more particularly DNA-binding proteins with a relatively high percentageof Lysine and Arginine residues (“Arg- and Lys-rich proteins”), can beused with the compositions of the invention. A DNA-binding protein canbe sequence-specific, partially sequence specific, or non-specific.

See U.S. Pat. No. 5,354,844 (Protein-polycation conjugates) to Beug, etal.; U.S. Pat. No. 5,972,900 (Delivery of nucleic acid to cells) toFerkol, Jr., et al.; U.S. Pat. No. 5,166,320 (Carrier system and methodfor the introduction of genes into mammalian cells); and U.S. Pat. Nos.6,008,336, 5,844,107 and 5,877,302 (Compacted nucleic acids and theirdelivery to cells), U.S. Pat. No. 6,077,835 (Delivery of compactednucleic acid to cells), all to Hanson, et al. U.S. Pat. No. 6,333,396 toFilpula, et al. (Method for targeted delivery of nucleic acids)describes a single-chain antigen-binding polypeptide comprising, at itsC-terminus, N-terminus, or both, basic amino acid residues selected fromthe group consisting of oligo-Lys, oligo-Arg and combinations thereof.U.S. Pat. No. 6,281,005 (Automated nucleic acid compaction device) toHanson, et al. describes a device that can be used to prepare compactedDNA complexes.

Non-Eukaryotic Histonelike Proteins

One class of DNA-binding, Arg- and Lys-rich proteins that can be used inthe invention is any non-eukaryotic histonelike protein. By way ofnon-limiting example, these include HU protein and IHF (integration hostfactor). HU and IHF proteins have been identified and cloned from avariety of eubacteria and archaea, including by way of non-limitingexample Aeromonas proteolytica, Bacillus caldolyticus, Bacilluscaldotenax, Bacillus cereus, Bacillus globigii, Bacillusstearothermophilus, Bacillus subtilis, Bifidobacterium longum, Borreliaburgdorferi, Campylobacter jejuni, Escherichia coli, Mycoplasmagallisepticum, Neisseria gonorrhoeae, Pseudomonas aeruginosa,Pseudomonas putida, Rhodobacter capsulatus, Salmonella typhimurium,Serratia marcescens, and Thermotoga maritima.

Histones

Another class of DNA-binding, Arg- and Lys-rich protein that can be usedin the complexes and compositions of the present invention is a histoneor mixture of a histones. Any histone protein, including withoutlimitation H1, H2A, H2B, H3 and H4, can be used. The use of histoneproteins is described in the following references, all of which areincorporated herein by reference in their entireties: Balicki D, BeutlerE. 1997. Histone H2A significantly enhances in vitro DNA transfection.Mol. Med. 3:782-787; Balicki et al. 2000. Histone H2A-mediated transientcytokine gene delivery induces efficient antitumor responses in murineneuroblastoma. Proc Natl Acad Sci USA 97:11500-11504; Balicki et al.2002. Structure and function correlation in histone H2A peptide-mediatedgene transfer. Proc Natl Acad Sci USA 99:7467-7471; Demirhan et al.1998. Histone-mediated transfer and expression of the HIV-1 tat gene inJurkat cells. J Hum Virol. 1:430-440; and Zaitsev et al. 2002. HistoneH1-mediated transfection: role of calcium in the cellular uptake andintracellular fate of H1-DNA complexes. Acta Histochem 104:85-92. Seealso U.S. Pat. Nos. 6,180,784 and 5,744,335 (both entitled “Process oftransfecting a cell with a polynucleotide mixed with an amphipathiccompound and a DNA-binding protein”), both to Wolff, et al.; U.S. Pat.No. 6,458,382 (“Nucleic acid transfer complexes”) to Herweijer, et al.;published PCT application WO 96/14424 (“DNA transfer method”) toHallybone; and published PCT application WO 99/19502, EP 0 967 288 A1,and EP 0 908 521 A1 (all entitled “Transfection System for the transferof nucleic acids into cells”), all to Chandra, et al.

The human histone-like protein described in U.S. Pat. Nos. 5,851,799,5,981,221 and 5,908,831 (all entitled “Histone-like protein), all toBandman, et al., and the protein and peptide sequences described in U.S.Pat. Nos. 5,945,400 and 6,200,956, and Published PCT application WO96/25508 (all entitled “Nucleic acid-containing composition, preparationand use thereof”), all to Scherman, et al., can also be used to practicethe invention. Chemically modified histone proteins, including by way ofnon-limiting example galactosylated histones (Chen, et al., Hum GeneTher 5:429-435, 1994), can be used in the invention.

Nucleic Acids

As noted above, the complexes of the present invention may comprise oneor more nucleic acids or nucleic acid molecules, which often willcomprise one or more genes of interest, that can be delivered to cells,tissues, organs or organisms using the compositions, complexes andmethods of the present invention. As used herein, the term “nucleicacids” (which is used herein interchangeably and equivalently with theterm “nucleic acid molecules”) refers to nucleic acids (including DNA,RNA, and DNA-RNA hybrid molecules) that are isolated from a naturalsource; that are prepared in vitro, using techniques such as PCRamplification or chemical synthesis; that are prepared in vivo, e.g.,via recombinant DNA technology; or that are prepared or obtained by anyappropriate method. Nucleic acids used in accordance with the inventionmay be of any shape (linear, circular, etc.) or topology(single-stranded, double-stranded, linear, circular, supercoiled,torsional, nicked, etc.). The term “nucleic acids” also includes withoutlimitation nucleic acid derivatives such as peptide nucleic acids (PNAS)and polypeptide-nucleic acid conjugates; nucleic acids having at leastone chemically modified sugar residue, backbone, internucleotidelinkage, base, nucleotide, nucleoside, or nucleotide analog orderivative; as well as nucleic acids having chemically modified 5′ or 3′ends; and nucleic acids having two or more of such modifications. Notall linkages in a nucleic acid need to be identical.

Examples of nucleic acids include without limitation oligonucleotides(including but not limited to antisense oligonucleotides, ribozymes andoligonucleotides useful in RNA interference (RNAi)), aptamers,polynucleotides, artificial chromosomes, cloning vectors and constructs,expression vectors and constructs, gene therapy vectors and constructs,rRNA, tRNA, mRNA, mtRNA, and DNA, and the like. For reviews of thelatter type of nucleic acid, see Muto A, Ushida C, Himeno H. A bacterialRNA that functions as both a tRNA and an mRNA. Trends Biochem Sci.23:25-29, 1998; and Gillet R, Felden B. Emerging views on tmRNA-mediatedprotein tagging and nibosome rescue. Mol. Microbiol. 42:879-885, 2001.

Oligonucleotides

As used in the present invention, an oligonucleotide is a synthetic orbiologically produced molecule comprising a covalently linked sequenceof nucleotides which may be joined by a phosphodiester bond between the3′ position of the pentose of one nucleotide and the 5′ position of thepentose of the adjacent nucleotide. As used herein, the term“oligonucleotide” includes natural nucleic acid molecules (i.e., DNA andRNA) as well as non-natural or derivative molecules such as peptidenucleic acids, phophothioate-containing nucleic acids,phosphonate-containing nucleic acids and the like. In addition,oligonucleotides of the present invention may contain modified ornon-naturally occurring sugar residues (e.g., arabinose) and/or modifiedbase residues. The term oligonucleotide encompasses derivative moleculessuch as nucleic acid molecules comprising various natural nucleotides,derivative nucleotides, modified nucleotides or combinations thereof.Oligonucleotides of the present invention may also comprise blockinggroups which prevent the interaction of the molecule with particularproteins, enzymes or substrates.

Oligonucleotides include without limitation RNA, DNA and hybrid RNA-DNAmolecules having sequences that have minimum lengths of e nucleotides,wherein “e” is any whole integer from about 2 to about 15, and maximumlengths of about f nucleotides, wherein ‘f’ is any whole integer fromabout 2 to about 200. In general, a minimum of about 6 nucleotides,preferably about 10, and more preferably about 12 to about 15nucleotides, is desirable to effect specific binding to a complementarynucleic acid strand.

In general, oligonucleotides may be single-stranded (ss) ordouble-stranded (ds) DNA or RNA, or conjugates (e.g., RNA moleculeshaving 5′ and 3′ DNA “clamps”) or hybrids (e.g., RNA:DNA pairedmolecules), or derivatives (chemically modified forms thereof).Single-stranded DNA is often preferred, as DNA is less susceptible tonuclease degradation than RNA. Similarly, chemical modifications thatenhance the specificity or stability of an oligonucleotide are preferredin some applications of the invention.

Certain types of oligonucleotides are of particular utility in thecompositions and complexes of the present invention, including but notlimited to antisense oligonucleotides, ribozymes, interfering RNAs andaptamers.

Antisense Oligonucleotides

Nucleic acid molecules suitable for use in the present invention includeantisense oligonucleotides. In general, antisense oligonucleotidescomprise nucleotide sequences sufficient in identity and number toeffect specific hybridization with a preselected nucleic acid. Antisenseoligonucleotides are generally designed to bind either directly to mRNAtranscribed from, or to a selected DNA portion of, a targeted gene,thereby modulating the amount of protein translated from the mRNA or theamount of mRNA transcribed from the gene, respectively. Antisenseoligonucleotides may be used as research tools, diagnostic aids, andtherapeutic agents.

Antisense oligonucleotides used in accordance with the present inventiontypically have sequences that are selected to be sufficientlycomplementary to the target mRNA sequence so that the antisenseoligonucleotide forms a stable hybrid with the mRNA and inhibits thetranslation of the mRNA sequence, preferably under physiologicalconditions. It is preferred but not necessary that the antisenseoligonucleotide be 100% complementary to a portion of the target genesequence. However, the present invention also encompasses the productionand use of antisense oligonucleotides with a different level ofcomplementarity to the target gene sequence, e.g., antisenseoligonucleotides that are at least about 50% complementary, at leastabout 55% complementary, at least about 60% complementary, at leastabout 65% complementary, at least about 70% complementary, at leastabout 75% complementary, at least about 80% complementary, at leastabout 85% complementary, at least about 90% complementary, at leastabout 91% complementary, at least about 92% complementary, at leastabout 93% complementary, at least about 94% complementary, at leastabout 95% complementary, at least about 96% complementary, at leastabout 97% complementary, at least about 98% complementary, or at leastabout 99% complementary, to the target gene sequence. In certainembodiments, the antisense oligonucleotide hybridizes to an isolatedtarget mRNA under the following conditions: blots are first incubated inprehybridization solution (5×SSC; 25 mM NaPO₄, pH 6.5; 1×Denhardt'ssolution; and 1% SDS) at 42° C. for at least 2 hours, and thenhybridized with radiolabelled cDNA probes or oligonucleotide probes(1×10⁶ cpm/ml of hybridization solution) in hybridization buffer (5×SSC;25 mM NaPO₄, pH 6.5; 1×Denhardt's solution; 250 ug/ml total RNA; 50%deionized formamide; 1% SDS; and 10% dextran sulfate). Hybridization for18 hours at 30-42° C. is followed by washing of the filter in 0.1-6×SSC,0.1% SDS three times at 25-55° C. The hybridization temperatures andstringency of the wash will be determined by the percentage of the GCcontent of the oligonucleotides in accord with the guidelines describedby Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd edition,1989, Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

Representative teachings regarding the synthesis, design, selection anduse of antisense oligonucleotides include without limitation U.S. Pat.No. 5,789,573, Antisense Inhibition of ICAM-1, E-Selectin, and CMVIE1/IE2, to Baker et al.; U.S. Pat. No. 6,197,584, Antisense Modulationof CD40 Expression, to Bennett et al.; and Ellington, 1992, CurrentProtocols in Molecular Biology, 2nd Ed., Ausubel et al., eds.; WileyInterscience, New York, Units 2.11 and 2.12.

Ribozymes

Nucleic acid molecules suitable for use in the present invention alsoinclude ribozymes. In general, ribozymes are RNA molecules havingenzymatic activities usually associated with cleavage, splicing orligation of nucleic acid sequences. The typical substrates for ribozymesare RNA molecules, although ribozymes may catalyze reactions in whichDNA molecules (or maybe even proteins) serve as substrates. Two distinctregions can be identified in a ribozyme: the binding region which givesthe ribozyme its specificity through hybridization to a specific nucleicacid sequence (and possibly also to specific proteins), and a catalyticregion which gives the ribozyme the activity of cleavage, ligation orsplicing. Ribozymes which are active intracellularly work in cis,catalyzing only a single turnover, and are usually self-modified duringthe reaction. However, ribozymes can be engineered to act in trans, in atruly catalytic manner, with a turnover greater than one and withoutbeing self-modified. Owing to the catalytic nature of the ribozyme, asingle ribozyme molecule cleaves many molecules of target RNA andtherefore therapeutic activity is achieved in relatively lowerconcentrations than those required in an antisense treatment (WO96/23569).

Representative teachings regarding the synthesis, design, selection anduse of ribozymes include without limitation U.S. Pat. No. 4,987,071, RNAribozyme polymerases, dephosphorylases, restriction endonbonucleases andmethods, to Cech et al.; and U.S. Pat. No. 5,877,021, B7-1 TargetedRibozymes, to Stinchcomb et al.; the disclosures of all of which areincorporated herein by reference in their entireties.

Nucleic Acids for RNAi (RNAi Molecules)

Nucleic acid molecules suitable for use in the present invention alsoinclude nucleic acid molecules, particularly oligonucleotides, useful inRNA interference (RNAi). In general, RNAi is one method for analyzinggene function in a sequence-specific manner. For reviews, see Tuschl,T., Chembiochem. 2:239-245 (2001), and Cullen, B. R., Nat. Immunol.3:597-599 (2002). RNA-mediated gene-specific silencing has beendescribed in a variety of model organisms, including nematodes (Parrish,S., et al., Mol Cell 6:1077-1087 (2000); Tabara, H., et al., Cell99:123-132 (1999); in plants, i.e., “co-suppression” (Napoli, C., etal., Plant Cell 2:279-289 (1990)) and post-transcriptional or homologousgene silencing (Hamilton, A. J. and D.C. Baulcombe, Science 286:950-952(1999); Hamilton, et al., EMBO J. 21:4671-4679 (2002)) (PTGS or HGS,respectively) in plants; and in fungi, i.e., “quelling” (Romano, N. andG. Macino, Mol Microbiol 6:3343-3353 (1992)). Examples of suitableinterfering RNAs include siRNAs, shRNAs and stRNAs. As one of ordinaryskill will readily appreciate, however, other RNA molecules havinganalogous interfering effects are also suitable for use in accordancewith this aspect of the present invention.

Small Interfering RNA (siRNA)

RNAi is mediated by double stranded RNA (dsRNA) molecules that havesequence-specific homology to their “target” mRNAs (Caplen, N. J., etal., Proc Natl Acad Sci USA 98:9742-9747 (2001)). Biochemical studies inDrosophila cell-free lysates indicates that the mediators ofRNA-dependent gene silencing are 21-25 nucleotide “small interfering”RNA duplexes (siRNAs). Accordingly, siRNA molecules are advantageouslyused in the compositions, complexes and methods of the presentinvention. The siRNAs are derived from the processing of dsRNA by anRNase known as Dicer (Bernstein, E., et al., Nature 409:363-366 (2001)).It appears that siRNA duplex products are recruited into a multi-proteinsiRNA complex termed RISC(RNA Induced Silencing Complex). Withoutwishing to be bound by any particular theory, it is believed that a RISCis guided to a target mRNA, where the siRNA duplex interactssequence-specifically to mediate cleavage in a catalytic fashion(Bernstein, E., et al., Nature 409:363-366 (2001); Boutla, A., et al.,Curr Biol 11:1776-1780 (2001); Hammond et al., 2000).

RNAi has been used to analyze gene function and to identify essentialgenes in mammalian cells (Elbashir, et al., Methods 26:199-213 (2002);Harborth, et al., J Cell Sci 114:4557-4565 (2001)), including by way ofnon-limiting example neurons (Krichevsky, A. M. and Kosik, K. S., ProcNatl Acad Sci USA 99:11926-11929 (2002)). RNAi is also being evaluatedfor therapeutic modalities, such as inhibiting or block the infection,replication and/or growth of viruses, including without limitationpoliovirus (Gitlin, et al, Nature 418:379-380 (2002)) and HIV (Capodici,et al., J Immunol 169:5196-5201 (2002)), and reducing expression ofoncogenes (e.g., the bcr-abl gene; Scherr, et al., Blood September 26(epub ahead of print) (2002)). RNAi has been used to modulate geneexpression in mammalian (mouse) and amphibian (Xenopus) embryos(Calegari, et al., Proc Natl Acad Sci USA 99:14236-14240 (2002), andZhou, et al., Nucleic Acids Res 30:1664-1669 (2002), respectively), andin postnatal mice (Lewis, et al., Nat Genet. 32:107-108 (2002)), and toreduce transgene expression in adult transgenic mice (McCaffrey, et al.,Nature 418:38-39 (2002)).

Molecules that mediate RNAi, including without limitation siRNA, can beproduced in vitro by chemical synthesis (Hohjoh, H., FEBS Lett521:195-199 (2002)), hydrolysis of dsRNA (Yang, et al., Proc Natl AcadSci USA 99:9942-9947 (2002)), by in vitro transcription with T7 RNApolymerase (Donze, 0. and Picard, D., Nucleic Acids Res 30:e46. (2002);Yu, et al., Proc Natl Acad Sci USA 99:6047-6052 (2002)), and byhydrolysis of double-stranded RNA using a nuclease such as E. coli RNaseIII (Yang, et al., Proc Natl Acad Sci USA 99:9942-9947 (2002)). RNAimolecules can also be expressed inside cells by endogenous RNApolymerases, using for example RNA Pol III which acts on the U6 RNApromoter (Yu, et al., Proc Natl Acad Sci USA 99:6047-6052 (2002); Paul,et al., Nat Biotechnol 20:505-508 (2002)). For example, the commerciallyavailable GeneSuppressor System (IMGENEX, San Diego, Calif.) usesvectors comprising the U6 promoter to generate RNAi molecules in vivo.Viral vectors for siRNA (Xia, et al., Nat Biotechnol 20:1006-1010(2002)) including, by way of non-limiting example, retroviruses (Devroe,E. and Silver, P. A., BMC Biotechnol 2:15 (2002)), have also beendescribed. Methods have been described for determining the efficacy andspecificity of siRNAs in cell culture and in vivo (Bertrand, et al.,Biochem Biophys Res Commun 296:1000-1004 (2002); Lassus, et al., SciSTKE 2002(147):PL13 (2002); Leirdal, M. and Sioud, M., Biochem BiophysRes Commun 295:744-748 (2002)).

Because the Dicer RNase facilitates siRNA production, it is expectedthat cells that express Dicer will demonstrate a quicker and/or morerobust response to dsRNA-mediated RNAi, and that cells that overexpressDicer will respond even more quickly and/or more robustly.Overexpression of Dicer may be achieved by cloning a gene for a Dicerprotein (e.g., the Drosophila DCR-1 gene), or orthologs or homologsthereof, into an expression vector or cassette that is placed into acell of choice. Examples of cloned DCR genes include without limitationhomologs and orthologs of DCR from mice (Nicholson, R H. and Nicholson,A. W., Mamm. Genome 13:67-73 (2002)), accession No. NM148948; humans(Nagase, T., et al., DNA Res. 6:63-70 (1999)), accession No. NM 030621;as well as the Drosophila Dicer-2 (DCR-2) gene (Adams, et al, Science287:2185-2195 (2000)), accession No. NM 079054.

In another embodiment, therapeutic nucleic acid molecules (e.g., siNAmolecules) delivered exogenously optimally are stable within cells untilreverse transcription of the RNA has been modulated long enough toreduce the levels of the RNA transcript. The nucleic acid molecules areresistant to nucleases in order to function as effective intracellulartherapeutic agents. Improvements in the chemical synthesis of nucleicacid molecules described in the instant invention and in the art haveexpanded the ability to modify nucleic acid molecules by introducingnucleotide modifications to enhance their nuclease stability asdescribed above.

In yet another embodiment, siNA molecules having chemical modificationsthat maintain or enhance enzymatic activity of proteins involved in RNAiare used. Such nucleic acids are also generally more resistant tonucleases than unmodified nucleic acids. Thus, in vitro and/or in vivothe activity should not be significantly lowered.

In one embodiment, nucleic acid molecules of the invention that act asmediators of the RNA interference gene silencing response aredouble-stranded nucleic acid molecules. In another embodiment, the siNAmolecules of the invention consist of duplexes containing about 19 basepairs between oligonucleotides comprising about 19 to about 25 (e.g.,about 19, 20, 21, 22, 23, 24 or 25) nucleotides. In yet anotherembodiment, siNA molecules of the invention comprise duplexes withoverhanging ends of about 1 to about 3 (e.g., about 1, 2, or 3)nucleotides, for example, about 21-nucleotide duplexes with about 19base pairs and 3′-terminal mononucleotide, dinucleotide, ortrinucleotide overhangs.

In one embodiment, a siNA molecule of the invention comprises modifiednucleotides while maintaining the ability to mediate RNAi. The modifiednucleotides can be used to improve in vitro or in vivo characteristicssuch as stability, activity, and/or bioavailability. For example, a siNAmolecule of the invention can comprise modified nucleotides as apercentage of the total number of nucleotides present in the siNAmolecule. As such, a siNA molecule of the invention can generallycomprise about 5% to about 100% modified nucleotides (e.g., 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or 100% modified nucleotides). The actual percentage ofmodified nucleotides present in a given siNA molecule will depend on thetotal number of nucleotides present in the siNA. If the siNA molecule issingle stranded, the percent modification can be based upon the totalnumber of nucleotides present in the single stranded siNA molecules.Likewise, if the siNA molecule is double stranded, the percentmodification can be based upon the total number of nucleotides presentin the sense strand, antisense strand, or both the sense and antisensestrands.

Short Hairpin RNAs (shRNAs)

Paddison, P. J., et al., Genes & Dev. 16:948-958 (2002) have used smallRNA molecules folded into hairpins as a means to effect RNAi.Accordingly, such short hairpin RNA (shRNA) molecules are alsoadvantageously used in the compositions, complexes and methods of thepresent invention. The length of the stem and loop of functional shRNAsvaries; stem lengths can range anywhere from about 25 to about 30 nt,and loop size can range between 4 to about 25 nt without affectingsilencing activity. While not wishing to be bound by any particulartheory, it is believed that these shRNAs resemble the dsRNA products ofthe Dicer RNase and, in any even, have the same capacity for inhibitingexpression of a specific gene.

In order to express siRNA and shRNA long-term in vivo for, by way ofnon-limiting example, gene therapy and developmental studies, plasmidsthat express these RNAs have been generated. Expression vectors thatcontinually express siRNAs in stably transfected mammalian cells havebeen developed. Other plasmids have been engineered to express smallhairpin RNAs (shRNAs) lacking poly (A) tails. Transcription of shRNAs isinitiated at a polymerase III (pol II) promoter and is believed to beterminated at position 2 of a 4-5-thymine transcription terminationsite. Upon expression, shRNAs are thought to fold into a stem-loopstructure with 3′ UU-overhangs. Subsequently, the ends of these shRNAsare processed, converting the shRNAs into .about.21 nt siRNA-likemolecules. The siRNA-like molecules can, in turn, bring aboutgene-specific silencing in the transfected cells, which may be, by wayof non-limiting example, mammalian or human cells.

Small Temporally Regulated RNAs (stRNAs)

Another group of small RNAs suitable for use in the compositions,complexes and methods of the present invention are the small temporallyregulated RNAs (stRNAs). In general, stRNAs comprise from about 20 toabout 30 nt (Banerjee and Slack, Control of development timing by smalltemporal RNAs: A paradigm for RNA-mediated regulation of geneexpression, Bioessays 24:119-129, 2002). Unlike siRNAs, stRNAsdownregulate expression of a target mRNA after the initiation oftranslation without degrading the mRNA.

Design and Synthesis of siRNA, shRNA, stRNA, Antisense and OtherOligonucleotides

One or more of the following guidelines may be used in designing thesequence of siRNA and other nucleic acids designed to bind to a targetmRNA, e.g., shRNA, stRNA, antisense oligonucleotides, ribozymes, and thelike, that are advantageously used in accordance with the presentinvention.

Nucleic acids that mediate RNAi may be synthesized in vitro usingmethods to produce oligonucleotides and other nucleic acids, as isdescribed elsewhere herein. In addition, dsRNA and other molecules thatmediate iRNA are available from commercial vendors, such as RibopharmaAG (Kulmach, Germany), Eurogentec (Seraing, Belgium) and Sequitur(Natick, Mass.). Eurogentec offers siRNA that has been labeled withfluorophores (e.g., HEX/TET; 5′Fluorescein, 6-FAM; 3′Fluorescein, 6-FAM;Fluorescein dT internal; 5′ TAMRA, Rhodamine; 3′ TAMRA, Rhodamine), andthese examples of fluorescent dsRNA that can be used in the invention.

Aptamers

Traditionally, techniques for detecting and purifying target moleculeshave used polypeptides, such as antibodies, that specifically bind suchtargets. Nucleic acids have long been known to specifically bind othernucleic acids (e.g., ones having complementary sequences). However,nucleic acids that bind non-nucleic target molecules have been describedand are generally referred to as aptamers. See, e.g., Blackwell, T. K.,et al., Science (1990) 250:1104-1110; Blackwell, T. K., et al., Science(1990) 250:1149-1152; Tuerk, C., and Gold, L., Science (1990)249:505-510; Joyce, G. F., Gene (1989) 82:83-87. Accordingly, nucleicacid molecules (e.g., oligonucleotides) suitable for use in the presentinvention also include aptamers. As applied to aptamers, the term“binding” specifically excludes the “Watson-Crick”-type bindinginteractions (i.e., A:T and G:C base-pairing) traditionally associatedwith the DNA double helix.

The term “aptamer” thus refers to a nucleic acid or a nucleic acidderivative that specifically binds to a target molecule, wherein thetarget molecule is either (i) not a nucleic acid, or (ii) a nucleic acidor structural element thereof that is bound by the aptatmer throughmechanisms other than duplex- or triplex-type base pairing.

In general, techniques for identifying aptamers involve incubating apreselected non-nucleic acid target molecule with mixtures (2 to 50members), pools (50 to 5,000 members) or libraries (50 or more members)of different nucleic acids that are potential aptamers under conditionsthat allow complexes of target molecules and aptamers to form. By“different nucleic acids” it is meant that the nucleotide sequence ofeach potential aptamer may be different from that of any other member,that is, the sequences of the potential aptamers are random with respectto each other. Randomness can be introduced in a variety of manners suchas, e.g., mutagenesis, which can be carried out in vivo by exposingcells harboring a nucleic acid with mutagenic agents, in vitro bychemical treatment of a nucleic acid, or in vitro by biochemicalreplication (e.g., PCR) that is deliberately allowed to proceed underconditions that reduce fidelity of replication process; randomizedchemical synthesis, i.e., by synthesizing a plurality of nucleic acidshaving a preselected sequence that, with regards to at least oneposition in the sequence, is random. By “random at a position in apreselected sequence” it is meant that a position in a sequence that isnormally synthesized as, e.g., as close to 100% A as possible (e.g.,5′-C-T-T-A-G-T-3′), is allowed to be randomly synthesized at thatposition (C-T-T-N-G-T, wherein N indicates a randomized position. At arandomized position, for example, the synthesizing reaction contains 25%each of A, T, C and G; or x % A, w % T, y % C and z % G, whereinx+w+y+z=100. The randomization at the position may be complete (i.e.,x=y=w=z=25%) or stoichastic (i.e., at least one of x, w, y and z is not25%).

In later stages of the process, the sequences are increasingly lessrandomized and consensus sequences may appear; in any event, it ispreferred to ultimately obtain an aptamer having a unique nucleotidesequence.

Aptamers and pools of aptamers are prepared, identified, characterizedand/or purified by any appropriate technique, including those utilizingin vitro synthesis, recombinant DNA techniques, PCR amplification, andthe like. After their formation, target:aptamer complexes are thenseparated from the uncomplexed members of the nucleic acid mixture, andthe nucleic acids that can be prepared from the complexes are candidateaptamers (at early stages of the technique, the aptamers generally beinga population of a multiplicity of nucleotide sequences having varyingdegrees of specificity for the target). The resulting aptamer (mixtureor pool) is then substituted for the starting apatamer (library or pool)in repeated iterations of this series of steps. When a limited number(e.g., a pool or mixture, preferably a mixture with less than 10members, most preferably 1) of nucleic acids having satisfactoryspecificity is obtained, the aptamer is sequenced and characterized.Pure preparations of a given aptamer are generated by any appropriatetechnique (e.g., PCR amplification, in vitro chemical synthesis, and thelike).

For example, Tuerk and Gold (Science (1990) 249:505-510) describe theuse of a procedure termed “systematic evolution of ligands byexponential enrichment” (SELEX). In this method, pools of nucleic acidmolecules that are randomized at specific positions are subjected toselection for binding to a nucleic acid-binding protein (see, e.g., PCTInternational Publication No. WO 91/19813 and U.S. Pat. No. 5,270,163).The oligonucleotides so obtained are sequenced and otherwisecharacterization. Kinzler, K. W., et al. (Nucleic Acids Res. (1989)17:3645-3653) used a similar technique to identify syntheticdouble-stranded DNA molecules that are specifically bound by DNA-bindingpolypeptides. Ellington, A. D., et al. (Nature (1990) 346: 818-822)describe the production of a large number of random sequence RNAmolecules and the selection and identification of those that bindspecifically to specific dyes such as Cibacron blue.

Another technique for identifying nucleic acids that bind non-nucleictarget molecules is the oligonucleotide combinatorial techniquedescribed by Ecker, D. J. et al. (Nuc. Acids Res. 21, 1853 (1993)) knownas “synthetic unrandomization of randomized fragments” (SURF), which isbased on repetitive synthesis and screening of increasingly simplifiedsets of oligonucleotide analogue libraries, pools and mixtures (Tuerk,C. and Gold, L. (Science 249, 505 (1990)). The starting library consistsof oligonucleotide analogues of defined length with one position in eachpool containing a known analogue and the remaining positions containingequimolar mixtures of all other analogues. With each round of synthesisand selection, the identity of at least one position of the oligomer isdetermined until the sequences of optimized nucleic acid ligand aptamersare discovered.

Once a particular candidate aptamer has been identified through a SURF,SELEX or any other technique, its nucleotide sequence can be determined(as is known in the art), and its three-dimensional molecular structurecan be examined by nuclear magnetic resonance (NMR). These techniquesare explained in relation to the determination of the three-dimensionalstructure of a nucleic acid ligand that binds thrombin in Padmanabhan,K. et al., J. Biol. Chem. 24, 17651 (1993); Wang, K. Y. et al.,Biochemistry 32, 1899 (1993); and Macaya, R. F. et al., Proc. Nat'l.Acad. Sci. USA 90, 3745 (1993). Selected aptamers may be resynthesizedusing one or more modified bases, sugars or backbone linkages. Aptamersconsist essentially of the minimum sequence of nucleic acid needed toconfer binding specificity, but may be extended on the 5′ end, the 3′end, or both, or may be otherwise derivatized or conjugated.

Oligonucleotide Synthesis

The oligonucleotides used in accordance with the present invention canbe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Other methods for such synthesis that are known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives. By way of non-limitingexample, see, e.g., U.S. Pat. No. 4,517,338 (Multiple reactor system andmethod for polynucleotide synthesis) to Urdea et al., and U.S. Pat. No.4,458,066 (Process for preparing polynucleotides) to Caruthers et al.;Lyer R P, Roland A, Zhou W, Ghosh K Modifiedoligonucleotides-synthesis-, properties and applications. Curr Opin Mol.Ther. 1:344-358, 1999; Verma S, Eckstein F. Modified oligonucleotides:synthesis and strategy for users. Annu Rev Biochem. 67:99-134, 1998;Pfleiderer W, Matysiak S, Bergmann F, Schnell R. Recent progress inoligonucleotide synthesis. Acta Biochim Pol. 43:37-44, 1996; Warren W J,Vella G. Principles and methods for the analysis and purification ofsynthetic deoxyribonucleotides by high-performance liquidchromatography. Mol. Biotechnol. 4:179-199, 1995; Sproat B S. Chemistryand applications of oligonucleotide analogues. J. Biotechnol.41:221-238, 1995; De Mesmaeker A, Altmann K H, Waldner A, Wendebom S.Backbone modifications in oligonucleotides and peptide nucleic acidsystems. Curr Opin Struct Biol. 5:343-355, 1995; Charubala R, PfleidererW. Chemical synthesis of 2′,5′-oligoadenylate analogues. Prog MolSubcell Biol. 14:114-138, 1994; Sonveaux E. Protecting groups inoligonucleotide synthesis. Methods Mol. Biol. 26:1-71, 1994; GoodchildJ. Conjugates of oligonucleotides and modified oligonucleotides: areview of their synthesis and properties. Bioconjug Chem. 1: 165-187,1990; Thuong N T. Asseline U. Chemical synthesis of natural and modifiedoligodeoxynucleotides. Biochimie. 67:673-684, 1985; Itakura K, Rossi JJ, Wallace R B. Synthesis and use of synthetic oligonucleotides. AnnuRev Biochem. 53:323-356, 1984; Caruthers M H, Beaucage S L, Becker C,Efcavitch J W, Fisher E F, Galluppi G, Goldman R, deHaseth P, MatteucciM, McBride L, et al. Deoxyoligonucleotide synthesis via thephosphoramidite method. Gene Amplif Anal. 3:1-26, 1983; Ohtsuka E,Ikehara M, Soll D. Recent developments in the chemical synthesis ofpolynucleotides. Nucleic Acids Res. 10:6553-6560, 1982; and Kossel H.Recent advances in polynucleotide synthesis. Fortschr Chem Org Naturst.32:297-508, 1975.

Oligonucleotides and other nucleic acids having accessory elements canalso be prepared for advantageous use in the compositions, complexes andmethods of the present invention. Some such accessory elements canspecifically bind or otherwise interact with another molecule for avariety of purposes, including without limitation:

Intracellular transport. For example, a nucleotide sequence thatlocalizes nucleic acids to mitochondria is described in U.S. Pat. No.5,569,754;

Cellular targeting. For example, the sequence of an aptamer that bindsto a cell surface molecule (e.g., a receptor, cellular adhesion protein,membrane lipid, etc.) can be included in order to direct theoligonucleotide complex to a particular type of cell;

Delivery of DNA-binding proteins. For example, a nucleotide sequencethat specifically binds a transcription factor can be included in orderto effect the delivery of the transcription factor at the same time asthe other components of the complex;

Delivery of recombination proteins. As an example, a site thatspecifically binds a recombination protein can be included. Therecombination protein can be a recombinase per se (e.g., lambdaintegrase and related site-specific recombinases) or a protein thatfacilitates or enhances recombination (e.g., a histonelike protein, suchas Integration Host Factor, IHF). In one embodiment, a histonelikeprotein (e.g., IHF) and a site-specific recombinase (e.g., lambdaintegrase or Xis) are incorporated into one or more complexes, and cellsare transfected therewith. The presence of IHF in transfected cellsincreases the amount of site-specific recombination mediated by theintegrase, thereby promoting recombination between specific sites (e.g.,attB, attP, attL, attR, etc.) on nucleic acids within the cells (Christet al., 2002. Site-specific recombination in eukaryotic cells mediatedby mutant lambda integrases: implications for synaptic complex formationand the reactivity of episomal DNA segments. J Mol Biol 319:305-314).Such cells include, without limitation, embryonic cells, such as stemcells (Christ N, Droge P. 2002. Genetic manipulation of mouse embryonicstem cells by mutant lambda integrase. Genesis 32:203-208). In anotherembodiment, mutants of lambda integrase that have activity in theabsence of IHF are used (Lorbach et al., 2000. Site-specificrecombination in human cells catalyzed by phage lambda integrasemutants. J Mol Biol 296:1175-81).

Chemical Modifications of Nucleic Acids

In certain embodiments, oligonucleotides used in accordance with thepresent invention may comprise one or more chemical modificationsincluding with neither limitation nor exclusivity base modifications,sugar modifications, and backbone modifications. In addition, a varietyof molecules can be conjugated to the oligonucleotides; see, e.g., thedescriptions of chemical conjunction of fluorophores to oligonucleotidesthat are present throughout the present disclosure. Other suitablemodifications include but are not limited to base modifications, sugarmodifications, backbone modifications, and the like.

Base Modifications

In certain embodiments, the oligonucleotides used in the presentinvention can comprise one or more base modifications. For example, thebase residues in aptamers may be other than naturally occurring bases(e.g., A, G, C, T, U, and the like). Derivatives of purines andpyrimidines are known in the art; an exemplary but not exhaustive listincludes aziridinylcytosine, 4-acetylcytosine, 5-fluorouracil,5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, inosine (and derivatives thereof),N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 7-methylguanine, 3-methylcytosine, 5-methylcytosine(5MC), N6-methyladenine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil-5-oxyaceticacid methylester, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid, and 2,6-diaminopurine. In addition to nucleicacids that incorporate one or more of such base derivatives, nucleicacids having nucleotide residues that are devoid of a purine or apyrimidine base may also be included in oligonucleotides and othernucleic acids.

Sugar Modifications

The oligonucleotides used in the present invention can also (oralternatively) comprise one or more sugar modifications. For example,the sugar residues in oligonucleotides and other nucleic acids may beother than conventional ribose and deoxyribose residues. By way ofnon-limiting example, substitution at the 2′-position of the furanoseresidue enhances nuclease stability. An exemplary, but not exhaustivelist, of modified sugar residues includes 2′ substituted sugars such as2′-O-methyl-, 2′-O-alkyl, 2′-O-alkyl, 2′-S-alkyl, 2′-S-alkyl,2′-fluoro-, 2′-halo, or 2′-azido-ribose, carbocyclic sugar analogs,alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and abasic nucleoside analogs such as methyl riboside, ethylriboside or propylriboside.

Backbone Modifications

The oligonucleotides used in the present invention can also (oralternatively) comprise one or more backbone modifications. For example,chemically modified backbones of oligonucleotides and other nucleicacids include, by way of non-limiting example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphos-photriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotri-esters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Chemicallymodified backbones that do not contain a phosphorus atom have backbonesthat are formed by short chain alkyl or cycloalkyl internucleosidelinkages, mixed heteroatom and alkyl or cycloalkyl internucleosidelinkages, or one or more short chain heteroatomic or heterocyclicinternucleoside linkages, including without limitation morpholinolinkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones;formacetyl and thioformacetyl backbones; methylene formacetyl andthioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; and amide backbones.

Vectors and Constructs

In certain embodiments, the nucleic acid molecules of the invention areprovided as vectors, particularly cloning vectors, expression vectors orgene therapy vectors. Vectors according to this aspect of the inventioncan be double-stranded or single-stranded and which may be DNA, RNA, orDNA/RNA hybrid molecules, in any conformation including but not limitedto linear, circular, coiled, supercoiled, torsional, nicked and thelike. These vectors of the invention include but are not limited toplasmid vectors and viral vectors, such as a bacteriophage, baculovirus,retrovirus, lentivirus, adenovirus, vaccinia virus, semliki forest virusand adeno-associated virus vectors, all of which are well-known and canbe purchased from commercial sources (Invitrogen; Carlsbad, Calif.;Promega, Madison Wis.; Stratagene, La Jolla Calif.).

In accordance with the invention, any vector may be used to constructthe cloning vectors and expression vectors of the invention. Inparticular, vectors known in the art and those commercially available(and variants or derivatives thereof) may in accordance with theinvention be engineered to include one or more recombination sites foruse in the methods of the invention. Such vectors may be obtained from,for example, Vector Laboratories Inc., Invitrogen, Promega, Novagen,NEB, Clontech, Boehringer Mannheim, Pharmacia, EpiCenter, OriGenesTechnologies Inc., Stratagene, Perkin Elmer, Pharmingen, ResearchGenetics. General classes of vectors of particular interest includeprokaryotic and/or eukaryotic cloning vectors, expression vectors,fusion vectors, two-hybrid or reverse two-hybrid vectors, shuttlevectors for use in different hosts, mutagenesis vectors, transcriptionvectors, vectors for receiving large inserts and the like. Other vectorsof interest include viral origin vectors (M13 vectors, bacterial phage λvectors, adenovirus vectors, and retrovirus vectors), high, low andadjustable copy number vectors, vectors which have compatible repliconsfor use in combination in a single host (pACYC184 and pBR322) andeukaryotic episomal replication vectors (pCDM8).

Particular vectors of interest include prokaryotic expression vectorssuch as pProEx-HT, pcDNA II, pSL301, pSE280, pSE380, pSE420, pTrcHisA,B, and C, pRSET A, B, and C (Invitrogen Corporation), pGEMEX-1, andpGEMEX-2 (Promega, Inc.), the pET vectors (Novagen, Inc.), pTrc99A,pKK223-3, the pGEX vectors, pEZZ18, pRIT2T, and pMC1871 (Pharmacia,Inc.), pKK233-2 and pKK388-1 (Clontech, Inc.), and variants andderivatives thereof. Vectors can also be made from eukaryotic expressionvectors such as pYES2, pAC360, pBlueBacHis A, B, and C, pVL1392,pBsueBacIII, pCDM8, pcDNA1, pZeoSV, pcDNA3 pREP4, pCEP4, pEBVHis,pFastBac, pFastBac HT, pFastBac DUAL, pSFV, and pTet-Splice(Invitrogen), pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2,pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCH110, andpKK232-8 (Pharmacia, Inc.), p3'SS, pXT1, pSG5, pPbac, pMbac, pMC1neo,and pOG44 (Stratagene, Inc.), and variants or derivatives thereof.

Other vectors of particular interest include pUC18, pUC19, pBlueScript,pSPORT, cosmids, phagemids, YACs (yeast artificial chromosomes), BACs(bacterial artificial chromosomes), MACs (mammalian artificialchromosomes), HACs (human artificial chromosomes), P1 (E. coli phage),pQE70, pQE60, pQE9 (Qiagen), pBS vectors, PhageScript vectors,BlueScript vectors, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene), pcDNA3,pSPORT1, pSPORT2, pCMVSPORT2.0 and pSV-SPORT1 (Invitrogen), pGEX,pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia), and variants or derivatives thereof.

Additional vectors of interest include pTrxFus, pThioHis, pLEX, pTrcHis,pTrcHis2, pRSET, pBlueBacHis2, pcDNA3.1/His, pcDNA3.1(−)/Myc-His,pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pAO815, pPICZ, pPICZa, pGAPZ,pGAPZa, pBlueBac4.5, pBlueBacHis2, pMelBac, pSinRep5, pSinHis, pIND,pIND(SP1), pVgRXR, pcDNA2.1. pYES2, pZErO1.1, pZErO-2.1, pCR-Blunt,pSE280, pSE380, pSE420, pVL1392, pVL1393, pCDM8, pcDNA1.1, pcDNA1.1/Amp,pcDNA3.1, pcDNA3.1/Zeo, pSe, SV2, pRc/CMV2, pRc/RSV, pREP4, pREP7,pREP8, pREP9, pREP10, pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni, andpCRBac from Invitrogen; λExCell, λgt11, pTrc99A, pKK223-3, pGEX-1λT,pGEX-2T, pGEX-2TK, pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-3X, pGEX-5X-1,pGEX-5X-2, pGEX-5X-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL, pMSG,pCH110, pKK232-8, pSL1180, pNEO, and pUC4K from Pharmacia;pSCREEN-1b(+), pT7Blue(R), pT7Blue-2, pCITE-4abc(+), pOCUS-2, pTAg,pET-32 LIC, pET-30 LIC, pBAC-2 cp LIC, pBACgus-2cp LIC, pT7Blue-2 LIC,pT7Blue-2, ASCREEN-1, λBlueSTAR, pET-3abcd, pET-7abc, pET9abcd,pET11abcd, pET12abc, pET-14b, pET-15b, pET-16b, pET-17b-pET-17xb,pET-19b, pET-20b(+), pET-21abed(+), pET-22b(+), pET-23abcd(+),pET-24abcd(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28abc(+),pET-29abc(+), pET-30abc(+), pET-31b(+), pET-32abc(+), pET-33b(+),pBAC-1, pBACgus-1, pBAC4x-1, pBACgus4x-1, pBAC-3 cp, pBACgus-2 cp,pBACsurf-1, plg, Signal plg, pYX, Selecta Vecta-Neo, Selecta Vecta-Hyg,and Selecta Vecta-Gpt from Novagen; pLexA, pB42AD, pGBT9, pAS2-1,pGAD424, pACT2, pGAD GL, pGAD GH, pGAD10, pGilda, pEZM3, pEGFP, pEGFP-1,pEGFP-N, pEGFP-C, pEBFP, pGFPuv, pGFP, p6xHis-GFP, pSEAP2-Basic,pSEAP2-Contral, pSEAP2-Promoter, pSEAP2-Enhancer, pβgal-Basic,pβgal-Control, pβgal-Promoter, pβgal-Enhancer, pCMVβ, pTet-Off, pTet-On,pTK-Hyg, pRetro-Off, pRetro-On, pIRES1neo, pIRES1hyg, pLXSN, pLNCX,pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo, pYEX 4T-1/2/3,pYEX-S1, pBacPAK-His, pBacPAK8/9, pAcUW31, BacPAK6, pTrip1Ex, λgt10,λgt11, pWE15, and λTrip1Ex from Clontech; Lambda ZAP II, pBK-CMV,pBK-RSV, pBluescript II KS.+−., pBluescript II SK.+−., pAD-GAL4,pBD-GAL4 Cam, pSurfscript, Lambda FIX II, Lambda DASH, Lambda EMBL3,Lambda EMBL4, SuperCos, pCR-Scrigt Amp, pCR-Script Cam, pCR-ScriptDirect, pBS.+−., pBC KS.+−., pBC SK.+−., Phagescript, pCAL-n-EK, pCAL-n,pCAL-c, pCAL-kc, pET-3abcd, pET-11abcd, pSPUTK, pESP-1, pCMVLacI,pOPRSVI/MCS, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pMC1neo PolyA, pOG44, pOG45, PFRTβGAL, pNEOβGAL, pRS403, pRS404, pRS405, pRS406,pRS413, pRS414, pRS415, and pRS416 from Stratagene.

Two-hybrid and reverse two-hybrid vectors of particular interest includepPC86, pDBLeu, pDBTrp, pPC97, p2.5, pGAD1-3, pGAD10, pACt, pACT2,pGADGL, pGADGH, pAS2-1, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA,pBD-GAL4, pHISi, pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5,pNLexA, pYESTrp and variants or derivatives thereof. Other suitablevectors will be readily apparent to the skilled artisan.

Cloning Vectors

Cloning vectors according to the invention include plasmids, cosmids,viral or phage DNA molecules or other DNA molecules that are capable ofautonomous replication in a host cell, via splicing of vector-bornenucleic acid into the genetic material (chromosomal or extrachromosomal)of the host cell without loss of an essential biological function of thevector, thereby facilitating the replication and cloning of the vector.The cloning vector may further contain a marker suitable for use in theidentification of cells transformed with the cloning vector. Markers maybe, for example, antibiotic resistance genes, e.g., tetracyclineresistance or ampicillin resistance. Clearly, methods of inserting adesired nucleic acid fragment which do not require the use of homologousrecombination, transpositions or restriction enzymes (such as, but notlimited to, UDG cloning of PCR fragments (U.S. Pat. No. 5,334,575,entirely incorporated herein by reference), T:A cloning, and the like)can also be applied to clone a fragment into a cloning vector to be usedaccording to the present invention. The cloning vector can furthercontain one or more selectable markers suitable for use in theidentification of cells transformed with the cloning vector.

Expression Vectors

Expression vectors according to the invention include vectors that arecapable of enhancing the expression of one or more genes that have beeninserted or cloned into the vector, upon transformation of the vectorinto a host. The cloned gene is usually placed under the control of(i.e., operably linked to) certain transcriptional regulatory sequencessuch as promoter sequences. In certain preferred embodiments in thisregard, the vectors provide for specific expression, which may beinducible and/or cell type-specific. Particularly preferred among suchvectors are those inducible by environmental factors that are easy tomanipulate, such as temperature and nutrient additives. Expressionvectors useful in the present invention include chromosomal-, episomal-and virus-derived vectors, e.g., vectors derived from bacterial plasmidsor bacteriophages, and vectors derived from combinations thereof, suchas cosmids and phagemids.

To produce expression vectors according to this aspect of the invention,one or more gene-containing nucleic acid molecules or oligonucleotideinserts should be operatively linked to an appropriate promoter in thevector (which may be provided by the vector itself (i.e., a “homologouspromoter”) or may be exogenous to the vector (i.e., a “heterologouspromoter), such as the phage lambda P_(L) promoter, the E. coli lac, trpand tac promoters, and the like. Other suitable promoters will be knownto the skilled artisan. The gene fusion constructs will further containsites for transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will preferablyinclude a translation initiation codon at the beginning, and atermination codon (UAA, UGA or UAG) appropriately positioned at the end,of the polynucleotide to be translated. The expression vectors alsopreferably include at least one selectable marker. Such markers includetetracycline or ampicillin resistance genes for culturing in E. coli andother bacteria.

Vectors, Compositions and Methods for Gene Therapy

In additional embodiments, the invention provides compositionscomprising one or more genetic constructs, including vectors (such asthe expression or cloning vectors described above), or one or more ofthe complexes of the invention, that may be useful in delivering nucleicacid molecules to cells, tissues, organs and organisms for therapeuticor prophylactic purposes. The invention further provides methods forpreparing nucleic acid molecules having regions of viral nucleic acids,as well as nucleic acid molecules prepared by such methods andcompositions comprising these nucleic acid molecules, useful for thenucleic acid delivery and therapeutic/prophylactic purposes describedabove and in more detail below.

In one embodiment, me present invention provides methods for treating orpreventing a physical disorder in an animal that is suffering from orpredisposed to the physical disorder, comprising introducing into theanimal one or more of the nucleic acid molecules, complexes orcompositions of the invention. According to the invention, an animal,particularly a mammal (preferably a human) that is suffering from, orthat is predisposed or susceptible to, a physical disorder may betreated by administering to the animal an effective dose of one or moreof the nucleic acid molecules, complexes or compositions of theinvention, optionally in combination with a pharmaceutically acceptablecarrier or excipient therefor. As used herein, an animal that is“suffering from” a particular physical disorder is defined as an animalthat exhibits one or more overt physical symptoms of the disorder thatare typically used in the diagnosis or identification of the disorderaccording to established medical and veterinary procedures and protocolsthat will be familiar to the ordinarily skilled artisan. Analogously, asused herein, an animal that is “predisposed to” or “susceptible to” aphysical disorder is defined as an animal that does not exhibit aplurality of overt physical symptoms of the disorder but that isgenetically, physiologically or otherwise at risk for developing thedisorder under appropriate physiological and environmental conditions.Hence, whether or not a particular animal is “suffering from,”“predisposed to” or “susceptible to” a particular physical disorder willbe apparent to the ordinarily skilled artisan upon determination of themedical history of the animal using methods that are routine in themedical and veterinary arts.

Physical disorders treatable or preventable with the compositions andmethods of the present invention include any physical disorder that maybe delayed, prevented, cured or otherwise treated by modulating immunesystem function, particularly activation and/or apoptosis inantigen-presenting cells, in an animal suffering from, or predisposed orsusceptible to, the physical disorder. Such physical disorders that maybe treatable or preventable using the compositions, complexes andmethods of the present invention include, but are not limited to,infectious diseases (particularly bacterial diseases (including withoutlimitation meningitis, pneumonia, tetanus, cholera, typhoid fever,staphylococcal skin infections, streptococcal pharyngitis, scarletfever, pertussis, diphtheria, tuberculosis, leprosy, rickettsialdiseases, bacteremia, bacterial venereal diseases and the like), viraldiseases (including without limitation meningitis, AIDS, influenza,rhinitis, hepatitis, polio, pneumonia, yellow fever, Lassa fever, Ebolafever and the like), and/or fungal diseases (including withoutlimitation cryptococcosis, blastomycosis, mucormycosis, histoplasmosis,aspergillosis, and the like), parasitic diseases (including withoutlimitation malaria, Leishmaniasis, filariasis, trypanasomiasis,schistosomiasis, and the like), cancers (such as carcinomas, melanomas,sarcomas, leukemias and the like), and other disorders treatable orpreventable using the methods and compositions of the present invention.Analogously, physical disorders that may be treatable or preventableusing the present compositions and methods include, but are not limitedto, immune system disorders (such as rheumatoid arthritis, multiplesclerosis, systemic lupus erythematosis, Crohn's Disease), and otherdisorders of analogous etiology. The compositions and methods of thepresent invention may also be used in the prevention of diseaseprogression, such as in chemoprevention of the progression of apremalignant lesion to a malignant lesion, and to treat an animalsuffering from, or predisposed to, other physical disorders that respondto treatment with compositions that activate, or inhibit/delay/preventor induce apoptosis in, antigen-presenting cells.

In a first, such aspect of the invention, the animal suffering from orpredisposed to a physical disorder may be treated by introducing intothe animal one or more of the nucleic acid molecules of the invention,optionally in the form of a vector and further optionally in the form ofa polypeptide-nucleic acid complex of the invention (or a composition ofthe invention comprising one or more such complexes). This approach,known generically as “gene therapy,” is designed to increase the levelof expression of a given gene, generally contained on the nucleic acidmolecule and/or in the administered complex, in the cells and/or tissuesof the animal, thereby inhibiting, delaying or preventing theprogression and/or development of the physical disorder, or to inducethe reversal, amelioration or remission of one or more overt symptoms orprocesses of the physical disorder. Analogous gene therapy approacheshave proven effective or to have promise in the treatment of a varietyof mammalian diseases such as cystic fibrosis (Drumm, M. L. et al., Cell62:1227-1233 (1990); Gregory, R. J. et al., Nature 347:358-363 (1990);Rich, D. P. et al., Nature 347:358-363 (1990)), Gaucher disease (Sorge,J. et al., Proc. Natl. Acad. Sci. USA 84:906-909 (1987); Fink, J. K etal., Proc. Natl. Acad. Sci. USA 87:2334-2338 (1990)), certain forms ofhemophilia (Bontempo, F. A. et al., Blood 69:1721-1724 (1987); Palmer,T. D. et al, Blood 73:438-445 (1989); Axelrod, J. H. et al., Proc. Natl.Acad. Sci. USA 87:5173-5177 (1990); Armentano, D. et al., Proc. Natl.Acad. Sci. USA 87:6141-6145 (1990)) and muscular dystrophy (Partridge,T. A. et al., Nature 337:176-179 (1989); Law, P. K. et al., Lancet336:114-115 (1990); Morgan, J. E. et al., J. Cell Biol. 111:2437-2449(1990)), and certain cancers such as metastatic melanoma (Rosenberg, S.A. et al., Science 233:1318-1321 (1986); Rosenberg, S. A. et al., N.Eng. J. Med. 319:1676-1680 (1988); Rosenberg, S. A. et al., N. Eng. J.Med. 323:570-578 (1990)).

In carrying out such gene therapy methods of the invention, a variety ofvectors, particularly viral vectors, are useful in forming the complexesand compositions of the invention. For example, adenoviruses areespecially attractive vehicles for delivering genes to or viarespiratory epithelia and the use of such vectors are included withinthe scope of the invention. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993), present a reviewof adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994), demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest 91:225-234 (1993); PCT PublicationNos. WO94/12649 and WO 96/17053; U.S. Pat. No. 5,998,205; and Wang etal., Gene Therapy 2:775-783 (1995), the disclosures of all of which areincorporated herein by reference in their entireties. Adeno-associatedviruses (AAV) and Herpes viruses, as well as vectors prepared from theseviruses have also been proposed for use in gene therapy (Walsh et al.,1993, Proc. Soc. Exp. Biol. Med. 204:289-300; U.S. Pat. No. 5,436,146;Wagstaff et al., Gene Ther. 5:1566-70 (1998)). Herpes viral vectors areparticularly useful for applications where gene expression is desired innerve cells.

In a preferred such approach, one or more nucleic acid and/or one ormore polypeptide of the present invention within the polymer complexesof the invention, is introduced into or administered to the animal thatis suffering from or predisposed to the physical disorder. Such nucleicacid molecules may be incorporated into a vector or virion suitable forintroducing the nucleic acid molecules into the cells or tissues of theanimal to be treated, to form a transfection vector. Suitable vectors orvirions for this purpose include those derived from retroviruses,adenoviruses, alphaviruses, herpes viruses and adeno-associated viruses.As one of ordinary skill will readily recognize, the complexes of theinvention also optionally may be combined with one or morepharmaceutically acceptable excipients or diluents to form apharmaceutical composition suitable for use in these methods of theinvention.

In addition, general methods for construction of gene therapy vectorsand the introduction thereof into affected animals for therapeuticpurposes may be obtained in the above-referenced publications, thedisclosures of which are specifically incorporated herein by referencein their entirety. In one such general method, vectors comprising thenucleic acid molecules of the present invention are directly introducedinto the cells or tissues of the affected animal, preferably byinjection, inhalation, ingestion or introduction into a mucous membranevia solution; such an approach is generally referred to as “in vivo”gene therapy. Alternatively, cells, tissues or organs, particularlythose containing one or more defective or nonfunctioning genes,containing pathological agents (e.g., bacteria, viruses, parasites,yeasts, etc.), or containing cancer cells or tumors, may be removed fromthe affected animal and placed into culture according to methods thatare well-known to one of ordinary skill in the art. The vectorscomprising the nucleic acid molecules of the invention, typicallycomprising one or more therapeutic genes or nucleic acid sequences, maythen be introduced into these cells or tissues by any of the methodsdescribed generally above for introducing oligonucleotides into a cellor tissue, and, after a sufficient amount of time to allow incorporationof the oligonucleotides, the cells or tissues may then be re-insertedinto the affected animal. Since the introduction of the therapeuticgenese or nucleic acid sequences is performed outside of the body of theaffected animal, this approach is generally referred to as “ex vivo”gene therapy.

For both in vivo and ex vivo gene therapy, the nucleic acid molecules(e.g., oligonucleotides) of the invention may alternatively beoperatively linked to a regulatory DNA sequence, which may be a promoteror an enhancer, or a heterologous regulatory DNA sequence such as apromoter or enhancer derived from a gene, cell or organism differentfrom that used as the source of the nucleic acid molecule being used ingene therapy, to form a genetic construct as described above. Thisgenetic construct may then be inserted into a vector, which is thendirectly introduced into the affected animal in an in vivo gene therapyapproach, or into the cells or tissues of the affected animal in an exvivo approach. In another embodiment, the genetic construct of theinvention may be introduced into the cells or tissues of the animal,either in vivo or ex vivo, in a molecular conjugate with a virus (e.g.,an adenovirus or an adeno-associated virus) or viral components (e.g.,viral capsid proteins; see WO 93/07283). In yet another embodiment, thegenetic construct of the invention may be introduced into the animal inthe form of a polypeptide-nucleic acid complex of the invention.Alternatively, transfected host cells, which may be homologous orheterologous, may be encapsulated within a semi-permeable barrier deviceand implanted into the affected animal, allowing passage of one or moretherapeutic polypeptides encoded by the nucleic acid molecules in theconjugate or complex of the invention into the tissues and circulationof the animal, but preventing contact between the animal's immune systemand the transfected cells (see WO 93/09222). These approaches result inincreased production of one or more therapeutic polypeptides by thetreated animal via (a) random insertion of the therapeutic gene(contained on the nucleic acid molecule of the invention) into the hostcell genome; or (b) incorporation of the therapeutic gene into thenucleus of the cells where it may exist as an extrachromosomal geneticelement. General descriptions of such methods and approaches to genetherapy may be found, for example, in U.S. Pat. No. 5,578,461; WO94/12650; and WO 93/09222; the disclosures of all of which areincorporated herein by reference in their entireties.

Release of Nucleic Acids Intracellularly

Once internalized into a cell (typically via endocytosis), transfectednucleic acids are usually sequestered within lipid membrane-enclosedvesicles (including endosomes, as well as components of the endoplasmicreticulum (ER) and/or Golgi apparatus). The release of nucleic acidsinto the cytosol from endosomes, the ER or the Golgi enhancestransfection. Endosomal disrupting agents can be used in the context ofthe invention and are defined herein as agents that cause or enhance therelease of nucleic acids into the cytosol. Endosomal disrupting agentscan act, by way of non-limiting example, by disrupting membranes ofendosomes, the ER, the Golgi apparatus and/or other membranes; blockingor reducing endosome fusion to lysosomes; and/or altering, preferablyraising, the pH of endosomes. The pH of an endosome is generally lowerthan that of the cytosol by one to two pH units. This pH gradient can beexploited for cellular delivery using agents that disrupt lipid bilayermembranes at pH 6.5 and below (Asokan A, Cho M J. 2002.

Exploitation of intracellular pH gradients in the cellular delivery ofmacromolecules. J Pharm Sci 91:903-913).

Membrane-disruptive pH-sensitive synthetic polymers have been describedand include by way of non-limiting example poly(amidoamine)s (PAAs)(Pattrick et al., 2001. Poly(amidoamine)-mediated intracytoplasmicdelivery of ricin A-chain and gelonin. J Control Release 77:225-32; U.S.Pat. No. 6,413,941); poly(propylacrylic acid) (PPAA) (Kyriakides et al.,2002. pH-sensitive polymers that enhance intracellular drug delivery invivo. J Control Release 78:295-303); and poly(ethyl acrylic acid)(PEAAc) (Murthy et al., 1999. The design and synthesis of polymers foreukaryotic membrane disruption. J Control Release 61:137-43).

Some cationic lipid transfection reagents, such as vectamidine andDMRIE-C, may have inherent endosomal disrupting properties. See EIOuahabi et al., 1997. The role of endosome destabilizing activity in thegene transfer process mediated by cationic lipids. FEBS Lett 414:187-92.Moreover, cationic lipids that are acid-labile have been described(Boomer et al., 2002. Formation of plasmid-based transfection complexeswith an acid-labile cationic lipid: characterization of in vitro and invivo gene transfer. Pharm Res 19:1292-1301; Wetzer et al., 2001.Reducible cationic lipids for gene transfer. Biochem J 356:747-756).

Other endosome disrupting agents include viral fusogenic peptides,including without limitation influenza virus hemagglutinin fusogenicpeptides (Bongartz et al., 1994. Improved biological activity ofantisense oligonucleotides conjugated to a fusogenic peptide. NucleicAcids Res 22:4681-4688) and synthetic derivatives thereof (Plank et al.,1994. The influence of endosome-disruptive peptides on gene transferusing synthetic virus-like gene transfer systems. J. Biol. Chem.269:12918-12924. These peptides are thought to change conformation atacidic pH and destabilize endosomal membranes.

The ricin A chain, which is capable of penetrating out of endosomes andinto the cytosol, can be attached to a nucleic acid or protein to inorder to effect the endosomal release thereof (Beaumell et al., 1993.ATP-dependent translocation of ricin across the membrane of purifiedendosomes J. Biol. Chem. 268:23661-23669).

Agents that alter the pH of endosomes can be used to practice theinvention. Lysosomotropic amines are generally thought to effect ofraising the pH of endosomes. Such agents include without limitationammonium chloride, 4-aminoquinolines (e.g., chloroquine, amodiaquine),8-aminoquinolines (e.g., primaquine and WR242511), pyrimethamine,quinacrine, quinine and quinidine (Tsiang H, Superti F. Ammoniumchloride and chloroquine inhibit rabies virus infection in neuroblastomacells. Brief report. Arch Virol 81:377-382; Deshpande et al., 1997.Efficacy of certain quinolines as pharmacological antagonists inbotulinum neurotoxin poisoning. Toxicon 35:433-445).

Artificial Chromosomes

The nucleic acid molecules used in the compositions, complexes andmethods of the present invention may alternatively be in the form ofartificial chromosomes (ACs). An AC is a DNA molecule that comprises, ata minimum, at least one origin of DNA replication (ori), one or moretelomeres and a centromere. Each ori is preferably derived from agenomic chromosome, so that replication of the AC is coordinated withcellular DNA replication. The telomeres are elements that preserve theterminal sequences of chromosomes for any number of rounds ofreplication and cell division. The centromere mediates propersegregation of the AC through each cell division (Willard H F.Centromeres: the missing link in the development of human artificialchromosomes. Curr Opin Genet Dev 8:219-225, 1998).

Ideally, ACs are stably maintained and are properly segregated duringboth mitosis and meiosis. Generally, an AC contains a segment of clonedDNA, and is usually more stable the larger the piece of cloned DNA. Itis possible to engineer ACs to improve or add functions (Grimes B, CookeH. Engineering mammalian chromosomes. Hum Mol Genet. 7:1635-1640, 1998;Saffery R, Choo K H. Strategies for engineering human chromosomes withtherapeutic potential. J Gene Med 4:5-13, 2002).

Bacterial and yeast artificial chromosomes (BACs and YACs, respectively)have been described. BACs and YACs are reviewed in Shizuya H,Kouros-Mehr H. The development and applications of the bacterialartificial chromosome cloning system. Keio J Med 50:26-30, 2001; andFabb S A, Ragoussis J. Yeast artificial chromosome vectors. Mol CellBiol Hum Dis Ser 5:104-124, 1995; Anand R. Yeast artificial chromosomes(YACs) and the analysis of complex genomes, Trends Biotechnol 10:35-40,1992.

Mammalian artificial chromosomes (MACs) have been prepared and may beused as vectors for somatic gene therapy. See Brown W R. Mammalianartificial chromosomes. Curr Opin Genet Dev 2:479-486, 1992; Huxley C.Mammalian artificial chromosomes and chromosome transgenics. TrendsGenet. 13:345-347, 1997; Ascenzioni F, Donini P, Lipps H J. Mammalianartificial chromosomes—vectors for somatic gene therapy. Cancer Lett118:135-142, 1997; Vos J M. Mammalian artificial chromosomes as toolsfor gene therapy. Curr Opin Genet Dev 8:351-359, 1998; and Vos j M.Therapeutic mammalian artificial episomal chromosomes. Curr Opin MolTher 1:204-215, 1999.

Human artificial chromosomes (HACs) have been described (Henning K A,Novotny E A, Compton S T, Guan X Y, Liu P P, Ashlock M A. Humanartificial chromosomes generated by modification of a yeast artificialchromosome containing both human alpha satellite and single-copy DNAsequences. Proc Natl Acad Sci USA. 96:592-597, 1999; Larin Z, Mejia J E.Advances in human artificial chromosome technology. Trends Genet.18:313-319, 2002). HACs include but are not limited to satelliteDNA-based artificial chromosomes (SATACs). SATACs have been made bymixing human telomeric DNA, genomic DNA, and arrays of repetitiveα-satellite DNA having centromeric activity (Hadlaczky G. SatelliteDNA-based artificial chromosomes for use in gene therapy. Curr Opin Mol.Ther. 3:125-132, 2001).

In addition to gene therapy, ACs have been used to stably clone largepieces of DNA in a variety of cell types (Schlessinger D, Nagaraja R.Impact and implications of yeast and human artificial chromosomes. AnnMed 30:186-191, 1998; Monaco A P, Larin Z. YACs, BACs, PACs and MACs:artificial chromosomes as research tools. Trends Biotechnol. 12:280-286,1994). In addition, ACs can be also be used in transgenic animaltechnologies to introduce large transgenes in animals, especially humantransgenes in mouse models of human genetic diseases. See Giraldo P,Montoliu L. Size matters: use of YACs, BACs and PACs in transgenicanimals. Transgenic Res 10:83-103, 2001; Jakobovits A, Lamb B T,Peterson K R. Production of transgenic mice with yeast artificialchromosomes. Methods Mol Biol 136:435-453, 2000; Lamb B T, Gearhart J D.YAC transgenics and the study of genetics and human disease. Curr OpinGenet Dev 5:342-348, 1995; Jakobovits A. YAC vectors. Humanizing themouse genome. Curr Biol 4:761-763, 1994; Huxley C. Transfer of YACs tomammalian cells and transgenic mice. Genet Eng (N Y) 16:65-91, 1994;Huxley C, Gnirke A. Transfer of yeast artificial chromosomes from yeastto mammalian cells. Bioessays 13:545-550, 1991; and Heintz N. BAC to thefuture: the use of bac transgenic mice for neuroscience research. NatRev Neurosci 2:861-870, 2001.

Peptide Nucleic Acids (PNAs)

The nucleic acid molecules used in the delivery compositions, complexesand methods of the present invention may alternatively be in the form ofpeptide nucleic acids (PNAs). PNAs are analogs of nucleic acid moleculesin which the backbone is a pseudopeptide rather than a sugar. Like DNAand RNA, a PNA molecule binds single-stranded nucleic acid having areverse complementary sequence; however, the neutral backbone of PNAscan result in stronger binding and greater specificity. For a review,see Corey D R. Peptide nucleic acids: expanding the scope of nucleicacid recognition. Trends Biotechnol 15:224-229, 1997. The synthesis ofPNAs is reviewed by Hyrup et al. (Peptide nucleic acids (PNA):synthesis, properties and potential applications. Bioorg Med. Chem.4:5-23, 1996). For exemplary protocols for making and using PNAs, seePeptide Nucleic Acids: Protocols and Applications, Nielsen, P. E. andEgholm, M., eds. Horizon Scientific Press, Norfolk, U.K. 1999. PNAs canbe prepared according to methods known in the art or purchasedcommercially from, e.g., Monomer Sciences Inc. (New Market, Ala., U.S.)and Dalton Chemical Laboratories Inc. (Toronto, ON, Canada). Methods forattaching fluorescent moieties to PNA have been described. See, e.g.,Murakami et al., A novel method for detecting HIV-1 by non-radioactivein situ hybridization: application of a peptide nucleic acid probe andcatalysed signal amplification. Pathol 194:130-135, 2001.

Fluorescent Molecules and Moieties

In certain embodiments, the compositions and polymer complexes of theinvention will comprise one or more marker or activation molecules ormoieties, such as one or more molecules or moieties that are linked to,complexed with, or comprise, one or more fluorophores. Contemplated bythis aspect of the invention are compositions in which the one or morefluorophores is linked (e.g., bound covalently or ionically) to one ormore components of the compositions of the invention (e.g.,fluorescently tagged nucleic acid molecules, nucleotides, proteins,peptides, and the like). Also contemplated by this aspect of theinvention are compositions in which the one or more fluorophores iscontained separately within the composition, without necessarily beingdirectly linked to one or more of the other components within thecomposition.

Fluorophores

For the purpose of the present invention, a fluorophore can be asubstance which itself fluoresces, or a substance that fluoresces inparticular situations (e.g., when in proximity to another fluorophore,as occurs in FRET). The term “fluorophore” or “fluor” is meant toencompass fluorescent moieties that are covalently linked to anothermolecule, fluorescent molecules that are non-covalently attached toanother molecule, as well as free fluorescent molecules. Molecules thatbecome fluorescent only after attachment to another molecule, such as apeptide or nucleic acid, are also within the scope of the invention.

In principal, any fluorophore now known, or later discovered, can beused in accordance with the methods, compositions and kits of thepresent invention. In certain embodiments, fluorophores suitable for usein the present invention include those that are excitable at, and/oremit fluorescence at, a wavelength falling within the range ofwavelengths from about 200 nm to about 800 nm; from about 250 nm toabout 800 nm; from about 250 nm to about 750 nm; from about 300 nm toabout 700 nm; from about 350 nm to about 650 nm; from about 400 nm toabout 600 nm; from about 450 nm to about 600 nm; from about 450 nm toabout 580 nm; from about 450 nm to about 575 nm; from about 450 nm toabout 570 nm; from about 500 nm to about 600 nm; from about 500 nm toabout 590 nm; from about 500 nm to about 580 nm; from about 500 nm toabout 575 mm; from about 500 nm to about 570 nm; and the like. As one ofordinary skill will readily appreciate, any fluorophore with anexcitation maximum and an emission maximum within the recited ranges issuitable for use in accordance with the present invention, whether ornot the actual, specific excitation and emission maxima for that givenfluorophore are specifically set forth above.

In view of the availability of an array of appropriate compounds, it iswell within the capabilities of one skilled in the art to choose areactive fluorescent molecule or set of molecules that is appropriate tothe practice of the present invention, given the above-noted guidelinesfor excitation and emission maxima. Many appropriate fluorophores arecommercially available from sources such as Molecular Probes Inc.(Eugene, Oreg.).

Many of these methods are quite appropriate for use in preparing thevarious compounds required to practice the present invention. Oneskilled in the art will be able, without undue experimentation, tochoose a suitable method for preparing a desired fluorescently labelednucleic acid, oligonucleotide or the like. See, for example, Protocolsfor Oligonucleotide Conjugates, Vol. 26 of Methods in Molecular Biology,Agrawal, ed., Humana Press, Totowa, N.J. (1994). Additionally, as theart of organic synthesis, particularly in the area of nucleic acidchemistry, continues to expand in scope new methods will be developedwhich are equally as suitable as those now known. The followingdiscussion is offered as representative of the array of compounds andtechniques that can be used to modify nucleic acids. Methods useful inconjunction with the present invention are not to be construed aslimited by this discussion.

Fluorescent moieties and molecules useful in practicing the presentinvention include but are not limited to derivatives of fluorescein,rhodamine, coumarin, dimethylaminonaph-thalene sulfonic acid (dansyl),pyrene, anthracene, nitrobenz-oxadiazole (NBD), acridine anddipyrrometheneboron difluoride. More specifically, non-limiting examplesof fluorescent moieties and molecules useful in practicing the presentinvention include, but are not limited to: carbocyanine, dicarbocyanine,merocyanine and other cyanine dyes (e.g., CyDye fluorophores, such asCy3, Cy3.5, Cy5, Cy5.5 and Cy7 from Pharmacia). These dyes have amaximum fluorescence at a variety of wavelengths: green (506 nm and 520nm), green-yellow (540 nm), orange (570 nm), scarlet (596 nm), far-red(670 nm), and near infrared (694 nm and 767 nm); coumarin and itsderivatives (e.g., 7-amino-4-methylcoumarin, aminocoumarin andhydroxycoumarin); BODIPY dyes (e.g., BODIPY FL, BODIPY 630/650, BODIPY650/665, BODIPY); fluorescein and its derivatives (e.g., fluoresceinisothiocyanate); rhodamine dyes (e.g. rhodamine green, rhodamine red,tetramethylrhodamine, rhodamine 6G and Lissamine rhodamine B); Alexadyes (e.g., Alexa Fluor-350, -430, -488, -532, -546, -568, -594, -663and -660, from Molecular Probes); fluorescent energy transfer dyes(e.g., thiazole orange-ethidium heterodimer, TOTAB, etc.); proteins withluminescent properties, e.g.: green fluorescent protein (GFP) andmutants and variants thereof, including by way of non-limiting examplefluorescent proteins having altered wavelengths (e.g., YFP, RFP, etc.).See Chiesa et al. (2001). Recombinant aequorin and green fluorescentprotein as valuable tools in the study of cell signalling. Biochem J.355:1-12; Sacchetti et al. (2000). The molecular determinants of theefficiency of green fluorescent protein mutants. Histol Histopathol.15:101-107; Larrick et al. (1995). Green fluorescent protein: untappedpotential in immunotechnology. Immunotechnology 1:83-86); aequorin andmutants and variants thereof; DsRed protein (Baird et al., 2000.Biochemistry, mutagenesis, and oligomerization of DsRed, a redfluorescent protein from coral. Proc Natl Acad Sci USA 97:11984-9), andmutants and variants thereof (see Verkhusha et al., 2001. An enhancedmutant of red fluorescent protein DsRed for double labeling anddevelopmental timer of neural fiber bundle formation. J Biol Chem276:29621-4; Bevis B J, Glick B S., 2002. Rapidly maturing variants ofthe Discosoma red fluorescent protein (DsRed). Nat Biotechnol 20:83-87;Terskikh et al., 2002. Analysis of DsRed Mutants. Space around thefluorophore accelerates fluorescence development. J Biol Chem277:7633-6; Campbell et al., 2002. A monomeric red fluorescent protein.Proc Natl Acad Sci USA 99:7877-82; and Knop et al., 2002. Improvedversion of the red fluorescent protein (drFP583/DsRed/RFP).Biotechniques 33:592, 594, 596-598); and other fluors, e.g., 6-FAM, HEX,TET, F12-dUTP, L5-dCTP, 8-anilino-1-napthalene sulfonate, pyrene,ethenoadenosine, ethidium bromide prollavine monosemicarbazide,p-terphenyl, 2,5-diphenyl-1,3,4-oxadiazole, 2,5-diphenyloxazole,p-bis[2-(5-phenyloxazolyl)]benzene,1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene, lanthanide chelates,Pacific blue, Cascade blue, Cascade Yellow, Oregon Green, Marina Blue,Texas Red, phycoerythrin, eosins and erytlhrosins; as well asderivatives of any of the preceding molecules and moieties.Fluorophores, and kits for attaching fluorophores to nucleic acids andpeptides, are commercially available from, e.g., Molecular Probes(Eugene, Oreg.) and Sigma/Aldrich (St. Louis, Mo.).

Fluorescent Oligonucleotides and Other Nucleic Acids

Fluorescent moieties useful in practicing the present invention can beattached to any location on a nucleic acid, including sites on the basesegment and sites on the sugar segment. Thus, the fluorophore iscovalently attached to a nucleic acid at a position selected from thegroup consisting of the 3′-terminus, the 5′-terminus, an internalposition and combinations thereof. See, generally, Goodchild, Bioconjug.Chem. 1: 165-187 (1990). Although any suitable fluorophore can beassociated with an oligonucleotide, some of the more commonly used onesare fluorescein, tetramethylrhodamine, Texas Red and Lissamine rhodamineB.

A number of techniques have been developed for converting specificconstituents of DNA and RNA strands into fluorescent adducts. For areview, see, Leonard and Tolman, in “Chemistry, Biology and ClinicalUses of Nucleoside Analogs,” A. Bloch, ed., Ann. N.Y. Acad. Sci.255:43-58 (1975).

Fluorescent G derivatives have also been prepared from the natural baseupon its reaction with variously substituted malondialdehydes. See,Leonard and Tolman, in “Chemistry, Biology and Clinical Uses ofNucleoside Analogs,” A. Bloch, ed., Ann. N.Y. Acad. Sci. 255:43-58(1975).

In addition to the various methods for converting the bases of an intactoligonucleotide into their fluorescent analogs, there are a number ofmethods for introducing fluorescence into an oligonucleotide during itsde novo synthesis.

Fluorescent Peptides, Polypeptides and Proteins

Fluorescent moieties useful in practicing the present invention can beattached to any location on a peptide or protein, including sites on theN-terminus, the C-terminus, a side group, an internal position andcombinations thereof.

By way of non-limiting example, a highly fluorescent molecule can bechemically linked to a native amino acid group. The chemicalmodification occurs on the amino acid side-chain, leaving the carboxyland amino functionalities free to participate in a polypeptide bondformation. Highly fluorescent dansyl chloride can be linked to thenucleophilic side chains of a variety of amino acids including lysine,arginine, tyrosine, cysteine, histidine, etc., mainly as a sulfonamidefor amino groups or sulfate bonds to yield fluorescent derivatives. Suchderivatization leaves the ability to form peptide bond intact, allowingfor the incorporation of dansyllysine into a protein.

More specifically, non-limiting examples of fluorescent moieties andmolecules useful in practicing the present invention includeamine-reactive fluorophores, which can react with the N-terminus of apeptide or a side group of an amino acid residue. These include withoutlimitation fluorophores associated with succinimidyl esters andcarboxylic acids thereof; aldehydes; sulfonyl chlorides, e.g., dansyl,pyrene, Lissamine rhodamine B and Texas Red derivatives; and arylatingreagents (e.g., NBD chloride, NBD fluoride and dichlorotriazines).

Fluorescamine is intrinsically nonfluorescent but reacts rapidly withprimary aliphatic amines, including those in peptides and proteins, toyield a blue-green-fluorescent derivative. The aromatic dialdehydeso-phthaldialdehyde (OPA) and naphthalene-2,3-dicarboxaldehyde (NDA) areessentially nonfluorescent until reacted with a primary amine to yield afluorescent isoindole. Sulfonyl chlorides, including dansyl chloride,1-pyrenesulfonyl chloride and dapoxyl sulfonyl chloride, react withamines to yield blue- or blue-green-fluorescent sulfonamides. FITC andbenzofuran isothiocyanates can be used. A unique method for specificderivatization of the N-terminus of peptides by FITC has been described(“Attachment of a single fluorescent label to peptides for determinationby capillary zone electrophoresis.” Zhao J Y, Waldron K C, Miller J,Zhang J Z, Harke H, Dovichi N J. J Chromatogr 608, 239-242, 1992).N-methylisatoic anhydride and the succinimidyl ester ofN-methylanthranilic acid can be used to prepare esters or amides of thesmall N-methylanthranilic acid fluorophore. The small size of thisfluorophore should reduce the likelihood that the label will interferewith the function of the protein.

The type of fluorophore, the site of its attachment to the peptide, thetype of linker used to attach the fluorophore and the site of attachmentof the peptide to the fluorophore can affect the efficiency of cellulardelivery and/or light-induced release of components from the complex.Specifically for fluorescein and fluorescein derivatives having the ringstructure of fluorescein, attachment of the peptide at the 5 ringposition of the fluorensein fluorophore is preferred.

Fluorophores can be linked to the peptide through linking groups whichcomprise a spacer portion and groups that form the covalent bonds to thepeptide and the fluorophore. For fluorescein and fluorescein derivativeshaving the ring structure of fluorescein, carboxy amine linkers arepreferred. Various reagents are commercially available for linkingfluorophores to peptides and for generating spacers in the linker.Spacers may include, for example, hydrocarbon spacers (—CH₂), ether orpolyether spacers.

Non-Covalent Association of Fluorophores with Nucleic Acids and Proteins

In one embodiment, the fluorophore is non-covalently bound to thetranslocating peptides and/or nucleic acids of the complexes. Withoutwishing to be limited to any particular theory, the association of atranslocating peptide and a nucleic acid is believed to be non-covalent.When the fluorophore is also non-covalently bound, to the peptide,nucleic acid, or both, the resulting complex is referred to as a fullynon-covalent complex.

Nucleic acids that bind fluorophores, including by way of non-limitingexample aptamers, can be prepared and used to prepare fully non-covalentcomplexes of nucleic acids, proteins and fluorophores. Similarly,proteins and peptides that bind fluorophores can be prepared, includingwithout limitation antibodies and derivatives thereof (e.g.,single-chain antibodies, camelid antibodies, CDRs, etc.).

A non-covalent specific binding pair can be used to prepare fullynon-covalent complexes. In this embodiment, one member of the specificbinding pair is associated with the nucleic acid or peptide, and theother member is associated with the fluorophore. The specific binding ofmembers of the pair to each other results in a non-covalent linkagebetween the nucleic acid or peptide that comprises a member of thebinding pair and the fluorophore. For example, biotin and streptavidincan be used to cause the non-covalent association of as fluorophore witha nucleic acid or protein. A strong non-covalent bond is formed betweenthe biotin and avidin moieties (the dissociation constant isapproximately 10¹⁵).

In one mode, a biotin moiety can be attached to the fluorophore, and thepeptide or oligonucleotide may comprise a streptavidin or avidin moiety.See Sano T, Vajda S, Cantor C R. Genetic engineering of streptavidin, aversatile affinity tag. J Chromatogr B Biomed Sci Appl. 715:85-91, 1998.For example, a fusion protein comprising VP22 translocating protein andstrepavidin may be generated and complexed with a biotinylatedfluorophore.

Compositions and Methods of Use

Thus, the invention provides polymer complexes comprising one or moreproteins or peptides, one or more nucleic acid molecules, and optionallyone or more fluorophores, produced by the methods of this invention andother methods known to those in the art, including automated andsemi-automated methods. For example, an automated device for formingcomplexes of nucleic acids and poly-Lys is described in U.S. Pat. No.6,281,005 to Casal, et al. In related aspects, the invention alsoprovides compositions comprising one or more such conjugates orcomplexes. Compositions according to this aspect of the invention willcomprise one or more (e.g., one, two, three, four, five, ten, etc.) ofthe above-described conjugates or complexes of the invention. In certainsuch aspects, the compositions may comprise one or more additionalcomponents, such as one or more buffer salts, one or more chaotropicagents, one or more detergents, one or more proteins (e.g., one or moreenzymes), one or more polymers and the like. The compositions of thisaspect of the invention may be in any form, including solid (e.g., drypowder) or solution particularly in the form of a physiologicallycompatible buffered salt solution comprising one or more of theconjugates of the invention).

Pharmaceutical Compositions

Certain compositions of the invention are particularly formulated foruse as pharmaceutical compositions for use in prophylactic, diagnosticor therapeutic applications. Such compositions will typically compriseone or more of the conjugates, complexes or compositions of theinvention and one or more pharmaceutically acceptable carriers orexcipients. The term “pharmaceutically acceptable carrier or excipient,”as used herein, refers to a non-toxic solid, semisolid or liquid filler,diluent, encapsulating material or formulation auxiliary of any typethat is capable of being tolerated by a recipient animal, including ahuman or other mammal, into which the pharmaceutical composition isintroduced, without adverse effects resulting from its addition.

The pharmaceutical compositions of the invention may be administered toa recipient via any suitable mode of administration, such as orally,rectally, parenterally, intrasystemically, vaginally, intraperitoneally,topically (as by powders, ointments, drops or transdermal patch),buccally, as an oral or nasal spray or by inhalation. The term“parenteral” as used herein refers to modes of administration thatinclude intravenous, intramuscular, intraperitoneal, intracisternal,subcutaneous and intra-articular injection and infusion.

Pharmaceutical compositions provided by the present invention forparenteral injection can comprise pharmaceutically acceptable sterileaqueous or nonaqueous solutions, dispersions, suspensions or emulsions,as well as sterile powders for reconstitution into sterile injectablesolutions or dispersions just prior to use. Examples of suitable aqueousand nonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (such as glycerol, propylene glycol, poly(ethyleneglycol), and the like), carboxymethylcellulose and suitable mixturesthereof, vegetable oils (such as olive oil), and injectable organicesters such as ethyl oleate. Proper fluidity can be maintained, forexample, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

Such pharmaceutical compositions of the present invention may alsocontain adjuvants such as preservatives, wetting agents, emulsifyingagents and dispersing agents. Prevention of the action of microorganismsmay be ensured by the inclusion of various antibacterial and antifungalagents, for example, paraben, benzyl alcohol, chlorobutanol, phenol,sorbic acid, and the like. It may also be desirable to include osmoticagents such as sugars, sodium chloride and the like. Prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents that delay absorption, such as aluminummonostearate, hydrogels and gelatin.

In some cases, in order to prolong the effect of the drugs, it isdesirable to slow the absorption from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor solubility in aqueous bodyfluids. The rate of absorption of the drug then depends upon its rate ofdissolution, which, in turn, may depend upon its physical form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of drug to carrier polymer and the nature ofthe particular carrier polymer employed, the rate of drug release can becontrolled. Examples of other biodegradable polymers includebiocompatible poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissues.

The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the activecompounds are mixed with at least one pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose, and gum acacia, c) humectants such as glycerol, d)disintegrating agents such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate,e) solution retarding agents such as paraffin, f) accelerators ofabsorption, such as quaternary ammonium compounds, g) wetting agentssuch as, for example, cetyl alcohol and glycerol monostearate, h)adsorbents such as kaolin and bentonite clay, and i) lubricants such astalc, calcium stearate, magnesium stearate, solid poly(ethyleneglycols), sodium lauryl sulfate, and mixtures thereof. In the case ofcapsules, tablets and pills, the dosage form may also comprise bufferingagents.

Solid compositions of a similar type may also be employed as fillers insoft- and hard-filled gelatin capsules using such excipients as lactose(milk sugar) as well as high molecular weight poly(ethylene glycols) andthe like. The solid dosage forms of tablets, dragees, capsules, pillsand granules can be prepared with coatings and shells such as enteric orchronomodulating coatings and other coatings well known in thepharmaceutical formulating art. They may optionally contain opacifyingagents and can also be of such a composition that they release theactive ingredient(s) only, or preferentially, in a certain part of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active compounds can also be in microencapsulated form, ifappropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirs. Inaddition to the active compounds, the liquid dosage forms may containinert diluents commonly used in the art such as, for example, water orother solvents, solubilizing agents and emulsifiers such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethyl formamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, poly(ethylene glycols) and fatty acid esters of sorbitan, andmixtures thereof. In addition to inert diluents, the oral compositionscan also include adjuvants such as wetting agents, emulsifying andsuspending agents, sweetening, flavoring and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar, and tragacanth, and mixturesthereof.

Topical administration includes administration to the skin or mucosa,including surfaces of the lung and eye. Compositions for topicaladministration, including those for inhalation, may be prepared as a drypowder which may be pressurized or non-pressurized. In non-pressurizedpowder compositions, the active ingredients in finely divided form maybe used in admixture with a larger-sized pharmaceutically acceptableinert carrier comprising particles having a size, for example, of up to100 micrometers in diameter. Suitable inert carriers include sugars suchas lactose and sucrose. Desirably, at least 95% by weight of theparticles of the active ingredient have an effective particle size inthe range of 0.01 to 10 micrometer.

Alternatively, the pharmaceutical composition may be pressurized andcontain a compressed gas, such as nitrogen or a liquefied gaspropellant. The liquefied propellant medium and indeed the totalcomposition may be preferably such that the active ingredients do notdissolve therein to any substantial extent. The pressurized compositionmay also contain a surface-active agent. The surface-active agent may bea liquid or solid non-ionic surface-active agent or may be a solidanionic surface-active agent. It is preferable to use the solid anionicsurface-active agent in the form of a sodium salt.

A further form of topical administration is to the eye. In this mode ofadministration, the conjugates or compositions of the invention aredelivered in a pharmaceutically acceptable ophthalmic vehicle, such thatthe active compounds are maintained in contact with the ocular surfacefor a sufficient time period to allow the compounds to penetrate theconjunctiva or the corneal and internal regions of the eye, as forexample the anterior chamber, posterior chamber, vitreous body, aqueoushumor, vitreous humor, cornea, iris/ciliary, lens, choroid/retina andsclera. The pharmaceutically acceptable ophthalmic vehicle may, forexample, be an ointment, vegetable oil or an encapsulating material.

Compositions for rectal or vaginal administration are preferablysuppositories that can be prepared by mixing the conjugates orcompositions of the invention with suitable non-irritating excipients orcarriers such as cocoa butter, PEG or a suppository wax, which are solidat room temperature but liquid at body temperature and therefore melt inthe rectum or vaginal cavity and release the drugs.

The pharmaceutical compositions used in the present therapeutic methodsmay also be administered in the form of liposomes. As is known in theart, liposomes are generally derived from phospholipids or other lipidsubstances. Liposomes are formed by mono- or multi-lamellar hydratedliquid crystals that are dispersed in an aqueous medium. Any non-toxic,physiologically acceptable and metabolizable lipid capable of formingliposomes can be used. In addition to one or more of the conjugates orcompositions of the invention, the present pharmaceutical compositionsin liposome form can also contain one or more stabilizers,preservatives, excipients, and the like. The preferred lipids are thephospholipids and the phosphatidyl cholines (lecithins), both naturaland synthetic. Methods to form liposomes are known in the art (see,e.g., Zalipsky, S., et al., U.S. Pat. No. 5,395,619). Liposomes thatcomprise phospholipids that are conjugated to poly(ethylene glycol)(“PEG”), most commonly phosphatidyl ethanolamine coupled tomonomethoxy-PEG, have advantageous properties, including prolongedlifetimes in the blood circulation of mammals (Fisher, D., U.S. Pat. No.6,132,763).

Other Uses

As noted elsewhere herein, the polymer, complexes and compositions ofthe present invention are advantageously used in methods for deliveringone or more components (e.g., one or more peptides and/or one or morenucleic acid molecules and/or one or more fluorophores) of the complexesand compositions to cells, tissues, organs or organisms. In particular,the invention provides controlled delivery of the one or more componentsof the complexes or compositions to cells, tissues, organs or organisms,thereby providing the user with the ability to regulate, temporally andspacially, the amount of a particular component that is released foractivity on the cells, tissues, organs or organisms.

In general, such methods of the invention involve one or moreactivities. For example, one such method of the invention comprises: (a)preparing one or more complexes or compositions of the invention asdetailed herein and (b) contacting one or more cells, tissues, organs ororganisms with the one or more complexes or compositions, underconditions favoring the uptake of the one or more complexes orcompositions of the invention by the cells, tissues, organs ororganisms. In another embodiment, the invention further provides for themethod comprising the added step (c), treating the cells, tissues,organs or organisms that contain the one or more complexes orcompositions of the invention with a treatment that releases one or moreof the bioactive components of the conjugates or compositions into thecells, tissues, organs or organisms.

Once the bioactive components of the complexes and/or compositions ofthe invention have entered the cells or been released into the cells,tissues, organs or organisms, the components proceed to carry out theirintended biological functions. For example, peptide components releasedinto the cells, tissues, organs or organisms may proceed to bind toreceptors or other compounds or components within the cells, tissues,organs or organisms; to participate in metabolic reactions within thecells, tissues, organs or organisms; to carry out, upregulate oractivate, or downregulate or inhibit, one or more enzymatic activitieswithin the cells, tissues, organs or organisms; to provide a missingstructural component to the cells, tissues, organs or organisms; toprovide one or more nutritional needs to the cells, tissues, organs ororganisms; to inhibit, treat, reverse or otherwise ameliorate one ormore processes or symptoms of a disease or physical disorder; and thelike. In other examples, nucleic acid components released into thecells, tissues, organs or organisms may proceed to bind to receptors orother compounds or components within the cells, tissues, organs ororganisms; to become incorporated into the genetic material within thecells, tissues, organs or organisms, whether chromosomal orextrachromosomal, genomic or otherwise; to carry out, upregulate oractivate, or downregulate or inhibit, one or more enzymatic activitieswithin the cells, tissues, organs or organisms; to provide a missinggenetic component to the cells, tissues, organs or organisms; toincrease or decrease the copy number of one or more genes within thecells, tissues, organs or organisms; to inhibit, treat, reverse orotherwise ameliorate one or more processes or symptoms of a disease orphysical disorder; and the like. In related aspects, the complexes andcompositions of the invention can be used to produce transgenic cells,tissues, organs or organisms, including non-human transgenic animalssuch as mice, rats, dogs, cows, pigs, rabbits, dogs, monkeys and thelike, using methods (such as nuclear transfer cloning) that arewell-known in the art and that will be familiar to the ordinarilyskilled artisan (see, e.g., U.S. Pat. Nos. 5,322,775, 5,366,894,5,476,995, 5,650,503 and 5,861,299; WIPO/PCT publication nos. WO98/37183 and WO 00/42174; U.S. patent application publication no.0012660-A1 (published on Jan. 31, 2002); Dai et al., NatureBiotechnology 20: 251-255 (2002); Betthauser et al, Nature Biotechnology18: 1055-1059 (2000); Onishi et al., Science 289:1188-1190 (2000); andPolejaeva et al., Nature 407:86-90 (2000). The disclosures of all ofthese documents are incorporated herein by reference in theirentireties).

Dose Regimens

The conjugates, complexes or compositions of the invention can beadministered in vitro, ex vivo or in vivo to cells, tissues, organs ororganisms to deliver one or more bioactive components (i.e., one or morepeptides or nucleic acid molecules) thereto. One of ordinary skill willappreciate that effective amounts of a given active compound, conjugate,complex or composition can be determined empirically and may be employedin pure form or, where such forms exist, in pharmaceutically acceptableformulation or prodrug form. The compounds, conjugates, complexes orcompositions of the invention may be administered to an animal(including a mammal, such as a human) patient in need thereof asveterinary or pharmaceutical compositions in combination with one ormore pharmaceutically acceptable excipients. It will be understood that,when administered to a human patient, the total daily, weekly or monthlyusage of the compounds and compositions of the present invention will bedecided by the attending physician within the scope of sound medicaljudgment. The therapeutically effective dose level for any particularpatient will depend upon a variety of factors including the type anddegree of the cellular response to be achieved; the identity and/oractivity of the specific compound(s), conjugate(s), complex(es) orcomposition(s) employed; the age, body weight or surface area, generalhealth, gender and diet of the patient; the time of administration,route of administration, and rate of excretion of the activecompound(s); the duration of the treatment, other drugs used incombination or coincidental with the specific compound(s), conjugate(s),complex(es) or composition(s); and like factors that are well known tothose of ordinary skill in the pharmaceutical and medical arts. Forexample, it is well within the skill of the art to start doses of agiven compound, conjugate, complex or composition of the invention atlevels lower than those required to achieve the desired therapeuticeffect and to gradually increase the dosages until the desired effect isachieved.

Dose regimens may also be arranged in a patient-specific manner toprovide a predetermined concentration of a given active compound in theblood, as determined by techniques accepted and routine in the art, e.g.size-exclusion, ion-exchange or reversed-phase HPLC. Thus, patient doseregimens may be adjusted to achieve relatively constant blood levels, asmeasured by HPLC, according to methods that are routine and familiar tothose of ordinary skill in the medical, pharmaceutical and/orpharmacological arts.

Diagnostic and Therapeutic Uses

In one embodiment, the diagnostic use of a polymer complex of thepresent invention is for locating an antigenic moiety, e.g., a cancer,within the body of an animal, especially a human, by administration of acomplex or composition of the invention, in which the complex orconjugate is labeled or comprises one or more detectable labels so as toenable detection, e.g. by optical, radiometric, fluorescent or resonantdetection according to art-known methods. Hence, in another aspect ofthe invention, the conjugates and compositions of the invention may beused in diagnostic or therapeutic methods, for example in diagnosing,treating or preventing a variety of physical disorders in an animal,particularly a mammal such as a human, predisposed to or suffering fromsuch a disorder. In such approaches, the goal of the therapy is to delayor prevent the development of the disorder, and/or to cure or induce aremission of the disorder, and/or to decrease or minimize the sideeffects of other therapeutic regimens. Hence, the complexes andcompositions of the present invention may be used for protection,suppression or treatment of physical disorders, such as infections ordiseases. The term “protection” from a physical disorder, as usedherein, encompasses “prevention,” “suppression” and “treatment”“Prevention” involves the administration of a complex or composition ofthe invention prior to the induction of the disease or physicaldisorder, while “suppression” involves the administration of the complexor composition prior to the clinical appearance of the disease; hence,“prevention” and “suppression” of a physical disorder typically areundertaken in an animal that is predisposed to or susceptible to thedisorder, but that is not yet suffering therefrom. “Treatment” of aphysical disorder, however, involves administration of the therapeuticcomplex or composition of the invention after the appearance of thedisease. It will be understood that in human and veterinary medicine, itis not always possible to distinguish between “preventing” and“suppressing” a physical disorder. In many cases, the ultimate inductiveevent or events may be unknown or latent, and neither the patient northe physician may be aware of the inductive event until well after itsoccurrence. Therefore, it is common to use the term “prophylaxis,” asdistinct from “treatment,” to encompass both “preventing” and“suppressing” as defined herein. The term “protection,” used inaccordance with the methods of the present invention, therefore is meantto include “prophylaxis.”

Methods according to this aspect of the invention may comprise one ormore steps that allow the clinician to achieve the above-describedtherapeutic goals. One such method of the invention may comprise, forexample: (a) identifying an animal (preferably a mammal, such as ahuman) suffering from or predisposed to a physical disorder, and (b)administering to the animal an effective amount of one or more of thepolymer complexes or compositions of the present invention as describedherein, particularly one or more complexes comprising one or morepeptides and/orone or more nucleic acids, and/or one or morefluorophores (or one or more pharmaceutical compositions comprising suchconjugates), such that the administration of the conjugate, complex orcomposition prevents, delays or diagnoses the development of, or curesor induces remission of, the physical disorder in the animal.

As used herein, an animal that is “predisposed to” a physical disorderis defined as an animal that does not exhibit a plurality of overtphysical symptoms of the disorder but that is genetically,physiologically or otherwise at risk for developing the disorder. In thepresent methods, the identification of an animal (such as a mammal,including a human) that is predisposed to, at risk for, or sufferingfrom a given physical disorder may be accomplished according to standardart-known methods that will be familiar to the ordinarily skilledclinician, including, for example, radiological assays, biochemicalassays (e.g., assays of the relative levels of particular peptides,proteins, electrolytes, etc., in a sample obtained from an animal),surgical methods, genetic screening, family history, physical palpation,pathological or histological tests (e.g., microscopic evaluation oftissue or bodily fluid samples or smears, immunological assays, etc.),testing of bodily fluids (e.g., blood, serum, plasma, cerebrospinalfluid, urine, saliva, semen and the like), imaging, (e.g., radiologic,fluorescent, optical, resonant (e.g., using nuclear magnetic resonance(NMR) or electron spin resonance (ESR)), etc. Once an animal has beenidentified by one or more such methods, the animal may be aggressivelyand/or proactively treated to prevent, suppress, delay or cure thephysical disorder.

Physical disorders that can be prevented, diagnosed or treated with thecomplexes, compositions and methods of the present invention include anyphysical disorders for which the peptide and/or nucleic acidcomponent(s) of the complexes or compositions may be used in theprevention, diagnosis or treatment. Such disorders include, but are notlimited to, a variety of cancers (e.g., breast cancers, uterine cancers,ovarian cancers, prostate cancers, testicular cancers, leukemias,lymphomas, lung cancers, neurological cancers, skin cancers, head andneck cancers, bone cancers, colon and other gastrointestinal cancers,pancreatic cancers, bladder cancers, kidney cancers and othercarcinomas, sarcomas, adenomas and myelomas); infectious diseases (e.g.,bacterial diseases, fungal diseases, viral diseases (including hepatitisand HIV/AIDS), parasitic diseases, and the like); genetic disorders(e.g., cystic fibrosis, amyotrophic lateral sclerosis, musculardystrophy, Gaucher's disease, Pompe's disease, severe combinedimmunodeficiency disorder and the like), anemia, neutropenia, hemophiliaand other blood disorders; neurological disorders (e.g., multiplesclerosis and Alzheimer's disease); enzymatic disorders (e.g., gout,uremia, hypercholesterolemia, and the like); disorders of uncertain ormultifocal etiology (e.g., cardiovascular disease, hypertension, and thelike); and other disorders of medical importance that will be readilyfamiliar to the ordinarily skilled artisan. The complexes, compositionsand methods of the present invention may also be used in the preventionof disease progression, such as in chemoprevention of the progression ofa premalignant lesion to a malignant lesion.

The therapeutic methods of the invention thus use one or moreconjugates, complexes or compositions of the invention, or one or moreof the pharmaceutical compositions of the invention, that may beadministered to an animal in need thereof by a variety of routes ofadministration, including orally, rectally, parenterally (includingintravenously, intramuscularly, intraperitoneally, intracisternally,subcutaneously and intra-articular injection and infusion),intrasystemically, vaginally, intraperitoneally, topically (as bypowders, ointments, drops or transdermal patch), buccally, as an oral ornasal spray or by inhalation. By the invention, an effective amount ofthe conjugates, complexes or compositions can be administered in vitro,ex vivo or in vivo to cells or to animals suffering from or predisposedto a particular disorder, thereby preventing, delaying, diagnosing ortreating the disorder in the animal. As used herein, “an effectiveamount of a conjugate (or complex or composition)” refers to an amountsuch that the conjugate (or complex or composition) carries out thebiological activity of the bioactive component (i.e., the peptide and/ornucleic acid component) of the conjugate/complex/composition, therebypreventing, delaying, diagnosing, treating or curing the physicaldisorder in the animal to which the conjugate, complex or composition ofthe invention has been administered. One of ordinary skill willappreciate that effective amounts of the conjugates, complexes orcompositions of the invention can be determined empirically, accordingto standard methods well-known to those of ordinary skill in thepharmaceutical and medical arts; see, e.g., Beers, M. H., et al., eds.(1999) Merck Manual of Diagnosis & Therapy, 17th edition, Merck and Co.,Rahway, N J; Hardman, J. G., et al., eds. (2001) Goodman and Gilman'sThe Pharmacological Basis of Therapeutics, 10th edition, McGraw-HillProfessional Publishing, Elnsford, N.Y.; Speight, T. M., et al., eds.(1997) Avery's Drug Treatment: Principles and Practice of ClinicalPharmacology and Therapeutics, 4th edition, Blackwell Science, Inc.,Boston; Katzung, B. G. (2000) Basic and Clinical Phamacology, 8thedition, Appleton and Lange, Norwalk Conn.; which references andreferences cited therein are incorporated entirely herein by reference.

It will be understood that, when administered to a human patient, thetotal daily, weekly or monthly dosage of the conjugates, complexes andcompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. For example,satisfactory results are obtained by administration of certain of theconjugates, complexes or compositions of the invention at appropriatedosages depending on the specific bioactive compound used, which dosageswill be readily familiar to the ordinarily skilled artisan or which maybe readily determined empirically using only routine experimentation.According to this aspect of the invention, the conjugates, complexes orcompositions can be administered once or, in divided doses, e.g., twiceper day or per week or per month. Appropriate dose regimens for variousmodes of administration (e.g., parenteral, subcutaneous, intramuscular,intraocular, intranasal, etc.) can also be readily determinedempirically, using only routine experimentation, or will be readilyapparent to the ordinarily skilled artisan, depending on the identity ofthe bioactive component (i.e., the peptide and/or nucleic acidcomponent) of the conjugate, complex or composition.

In additional applications, the conjugates, complexes and compositionsof the invention may be used to specifically target a diagnostic ortherapeutic agent to a cell, tissue, organ or organism that expresses areceptor for, binds, incorporates or otherwise can take up, thebioactive component (i.e., the peptide and/or nucleic acid component) ofthe conjugate, complex or composition. Methods according to this aspectof the invention may comprise, for example, contacting the cell, tissue,organ or organism with one or more conjugates, complexes or compositionsof the invention, which additionally comprise one or more diagnostic ortherapeutic agents, such that the conjugate, complex or composition istaken up by the cell, tissue, organ or organism by any mechanism (e.g.,by receptor-mediated endocytosis, pinocytosis, phagocytosis, diffusion,etc.), thereby delivering the diagnostic or therapeutic agent to thecell, tissue, organ or organism. The diagnostic or therapeutic agentused in accordance with this aspect of the invention may be, but is notlimited to, at least one agent selected from a nucleic acid, an organiccompound, a protein or peptide, an antibody, an enzyme, a glycoprotein,a lipoprotein, an element, a lipid, a saccharide, an isotope, acarbohydrate, an imaging agent, a detectable probe, or any combinationthereof, which may be detectably labeled as described herein. Atherapeutic agent used in this aspect of the present invention may havea therapeutic effect on the target cell (or tissue, organ or organism),the effect being selected from, but not limited to, correcting adefective gene or protein, a drug action, a toxic effect, a growthstimulating effect, a growth inhibiting effect, a metabolic effect, acatabolic affect, an anabolic effect, an antiviral effect, an antifungaleffect, an antibacterial effect, a hormonal effect, a neurohumoraleffect, a cell differentiation stimulatory effect, a celldifferentiation inhibitory effect, a neuromodulatory effect, ananti-neoplastic effect, an anti-tumor effect, an insulin stimulating orinhibiting effect, a bone marrow stimulating effect, a pluripotent stemcell stimulating effect, an immune system stimulating effect, and anyother known therapeutic effect that may be provided by a therapeuticagent delivered to a cell (or tissue, organ or organism) via a deliverysystem according to this aspect of the present invention.

Such additional therapeutic agents may be selected from, but are notlimited to, known and new compounds and compositions includingantibiotics, steroids, cytotoxic agents, vasoactive drugs, antibodiesand other therapeutic agents. Non-limiting examples of such agentsinclude antibiotics and other drugs used in the treatment of bacterialshock, such as gentamycin, tobramycin, nafcillin, parenteralcephalosporins, etc.; adrenal corticosteroids and analogs thereof, suchas dexamethasone, mitigate the cellular injury caused by endotoxins;vasoactive drugs, such as an alpha adrenergic receptor blocking agent(e.g., phenoxybenzamine), a beta adrenergic receptor agonist (e.g.,isoproterenol), and dopamine.

The conjugates, complexes and compositions of the invention may also beused for diagnosis of disease and to monitor therapeutic response. Incertain such methods, the conjugates, complexes or compositions of theinvention may comprise one or more detectable labels (such as thosedescribed elsewhere herein). In specific such methods, these detectablylabeled conjugates, complexes or compositions of the invention may beused to detect cells, tissues, organs or organisms expressing receptorsfor, or otherwise taking up, the bioactive component (i.e., the peptideand/or nucleic acid component) of the conjugates, complexes orcompositions. In one example of such a method, the cell, tissue, organor organism is contacted with one or more of the conjugates, complexesor compositions of the invention under conditions that favor the uptakeof the conjugate by the cell, tissue or organism (e.g., by binding ofthe conjugate to a cell-surface receptor or by pinocytosis or diffusionof the conjugate into the cell), and then detecting the conjugate boundto or incorporated into the cell using detection means specific to thelabel used (e.g., fluorescence detection for fluorescently labeledconjugates; magnetic resonance imaging for magnetically labeledconjugates; radioimaging for radiolabeled conjugates; etc.). Other usesof such detectably labeled conjugates may include, for example, imaginga cell, tissue, organ or organism, or the internal structure of ananimal (including a human), by administering an effective amount of alabeled form of one or more of the conjugates of the invention andmeasuring detectable radiation associated with the cell, tissue, organor organism (or animal). Methods of detecting various types of labelsand their uses in diagnostic and therapeutic imaging are well known tothe ordinarily skilled artisan, and are described elsewhere herein.

In another aspect, the conjugates and compositions of the invention maybe used in methods to modulate the concentration or activity of aspecific receptor for the bioactive component of the conjugate on thesurface of a cell that expresses such a receptor. By “modulating” theactivity of a given receptor is meant that the conjugate, upon bindingto the receptor, either activates or inhibits the physiological activity(e.g., the intracellular signaling cascade) mediated through thatreceptor. While not intending to be bound by any particular mechanisticexplanation for the regulatory activity of the conjugates of the presentinvention, such conjugates can antagonize the physiological activity ofa cellular receptor by binding to the receptor via the bioactivecomponent of the conjugate, thereby blocking the binding of the naturalagonist (e.g., the unconjugated bioactive component) and preventingactivation of the receptor by the natural agonist, while not inducing asubstantial activation of the physiological activity of the receptoritself. Methods according to this aspect of the invention may compriseone or more steps, for example contacting the cell (which may be done invitro or in vivo) with one or more of the conjugates of the invention,under conditions such that the conjugate (i.e., the bioactive componentportion of the conjugate) binds to a receptor for the bioactivecomponent on the cell surface but does not substantially activate thereceptor. Such methods will be useful in a variety of diagnostic, andtherapeutic applications, as the ordinarily skilled artisan will readilyappreciate.

Kits

The invention also provides kits comprising the polymers, conjugatesand/or compositions of the invention. Such kits typically comprise acarrier, such as a box, carton, tube or the like, having in closeconfinement therein one or more containers, such as vials, tubes,ampules, bottles and the like, wherein a first container contains one ormore of the conjugates and/or compositions of the present invention. Thekits encompassed by this aspect of the present invention may furthercomprise one or more additional components (e.g., reagents andcompounds) necessary for carrying out one or more particularapplications of the conjugates and compositions of the presentinvention, such as one or more components useful for the diagnosis,treatment or prevention of a particular disease or physical disorder(e.g., one or more additional therapeutic compounds or compositions, oneor more diagnostic reagents, one or more carriers or excipients, and thelike), one or more additional conjugates or compositions of theinvention, one or more sets of instructions, and the like.

Cells that may be transfected by a transfection polymer complexincorporating a nucleic acid of the invention include, for example,endothelial or epithelial cells, for example, cells of the any part ofthe airway epithelium, including bronchial and lung epithelium, and thecorneal endothelium. The airway epithelium is an important target forgene therapy for cystic fibrosis and asthma.

A transfection polymer complex as described above may be produced byadmixing the polymers and nucleic acid components. The individualcomponents of a transfection mixture of the invention are each asdescribed above in relation to the transfection polymer complex. Thepreferred components, preferred combinations of components, preferredratios of components and preferred order of mixing, both with regard tothe mixture and to the production of a polymer complex, are as describedabove in relation to the transfection polymer complex.

The present invention also provides a process for expressing a nucleicacid in host cells, which comprises contacting the host cells in vitroor in vivo with a receptor-targeted polymer complex of the inventioncomprising the nucleic acid and then culturing the host cells underconditions that enable the cells to express the nucleic acid.

The present invention further provides a process for the production of aprotein in host cells, which comprises contacting the host cells invitro or in vivo with a receptor-targeted polymer complex of theinvention that comprises a nucleic acid that encodes the protein,allowing the cells to express the protein, and obtaining the protein.The protein may be obtained either from the host cell or from theculture medium.

The present invention further provides a method of transfecting cellscomprising subjecting the cells to a polymer complex according to theinvention. The invention further provides cells, transfected with anucleic acid by a method according to the invention, and also theprogeny of such cells.

The present invention further provides a disease model for use intesting candidate pharmaceutical agent, which comprises cellstransfected by a method according to the invention with a nucleic acidsuitable for creating the disease model.

The present invention also provides a pharmaceutical composition whichcomprises a receptor-targeted polymer complex of the inventioncomprising a nucleic acid in admixture or conjunction with apharmaceutically suitable carrier. The composition may be a vaccine.

The present invention also provides a method for the treatment orprophylaxis of a condition caused in a human or in a non-human animal bya defect and/or a deficiency in a gene, which comprises administering tothe human or to the non-human animal a receptor-targeted polymer complexof the invention comprising a nucleic acid suitable for correcting thedefect or deficiency.

The present invention also provides a method for therapeutic orprophylactic immunization of a human or of a non-human animal, whichcomprises administering to the human or to the non-human animal areceptor-targeted polymer complex of the invention comprising anappropriate nucleic acid.

The present invention also provides a method of anti-sense therapy of ahuman or of a non-human animal, comprising anti-sense DNA administeringto the human or to the non-human animal a receptor-targeted polymercomplex of the invention comprising the anti-sense nucleic acid.

The present invention also provides the use of a receptor-targetedpolymer complex of the invention comprising a nucleic acid for themanufacture of a medicament for the prophylaxis of a condition caused ina human or in a non-human animal by a defect and/or a deficiency in agene, for therapeutic or prophylactic immunixation of a human or of anon-human animal, or for anti-sense therapy of a human or of a non-humananimal.

A non-human animal is, for example, a mammal, bird or fish, and isparticularly a commercially reared animal.

The treatments and uses described above may be carried out byadministering the respective delivery complex, agent or medicament in anappropriate manner, for example, administration may be topical, forexample, in the case of airway epithelia.

In a further embodiment, the present invention provides a kit comprisinga polymer complex of the invention comprising a nucleic acid. Thepresent invention also provides a kit that comprises the followingitems: (a) a polamide polymer; and (b) a nucleic acid. Such a nucleicacid may be single-stranded or double stranded and may be a plasmid oran artificial chromosome. The nucleic acid component may be provided bya vector complex suitable for the expression of the nucleic acid, thevector complex being either empty or comprising the nucleic acid. For invitro purposes, the nucleic acid may be a reporter gene. For in vivotreatment purposes, the nucleic acid may comprise DNA appropriate forthe correction or supplementation being carried out. Such DNA may be agene, including any suitable control elements, or it may be a nucleicacid with homologous recombination sequences. The components may beprovided individually or as complexes in salt free buffer (for examplein water, or 5% dextrose).

A kit generally comprises instructions, which preferably indicate thepreferred ratios of the components and the preferred order of use oradmixing of the components, for example, as described above. A kit maybe used for gene therapy, gene vaccination or anti-sense therapy.Alternatively, it may be used for transfecting a host cell with anucleic acid encoding a commercially useful protein i.e. to produce aso-called “cell factory”.

In a kit of the invention the components including the preferredcomponents are, for example, as described above in relation to adelivery complex of the present invention. The polycationic nucleic acidbinding component is preferably a polymer, as described above. Therations between the components are preferably as described above, as isthe order of mixing of the components.

Targets for gene therapy are well known and include monogenic disorders,for example, cystic fibrosis, various cancers, and infections, forexample, viral infections, for example, with HIV. For example,transfection with the p53 gene offers great potential for cancertreatment. Targets for gene vaccination are also well known, and includevaccination against pathogens for which vaccines derived from naturalsources are too dangerous for human use and recombinant vaccines are notalways effective, for example, hepatitis B virus, HIV, HCV and herpessimplex virus. Targets for anti-sense therapy are also known. Furthertargets for gene therapy and anti-sense therapy are being proposed asknowledge of the genetic basis of disease increases, as are furthertargets for gene vaccination. The present invention enhances thetransfection efficiency and hence the effectiveness of the treatment.

Delivery complexes of the invention may be effective for intracellulartransport of very large DNA molecules, for example, DNA larger than 125kb, which is particularly difficult using conventional vectors. Thisenables the introduction of artificial chromosomes into cells.

Transfection of the airways, for example, the bronchial epitheliumdemonstrates utility for gene therapy of, for example, respiratorydiseases, such as cystic fibrosis, emphysema, asthma, pulmonaryfibrosis, pulmonary hypertension and lung cancer.

Cystic fibrosis (CF) is the most common monogenic disorder in theCaucasian population. Morbidity is mainly associated with lung disease.CF is caused by mutations in the gene encoding the cystic fibrosistransmembrane conductance regulator protein (CFTR), a cell membranechannel that mediates secretion of chloride ions.

The enhanced levels of transfection make the method of the inventionparticularly suitable for the production of host cells capable ofproducing a desired protein, so-called “cell factories”. For long-termproduction, it is desirable that the introduced nucleic acid isincorporated in the genome of the host cell, or otherwise stablymaintained. That can be readily ascertained. As indicated above, therange of proteins produced in this way is large, including enzymes forscientific and industrial use, proteins for use in therapy andprophylaxis, immunogens for use in vaccines and antigens for use indiagnosis.

Accordingly, the present invention provides a method of testing drugs ina tissue model for a disease, wherein the tissue model comprisestransgenic cells obtained by transfecting cells with a nucleic acid bycontacting the cell with a receptor-targeted vector complex of theinvention comprising a nucleic acid.

In another embodiment, the present invention provides for the use forpolymers to deliver a concatemer to a cell. In another embodiment, thepresent invention provides for the use for polyamides to deliver aconcatemerized double-stranded oligonucleotide molecules (CODN) fortranscription factor decoys. In one embodiment, the concatemers consistof a variable number of end-to-end repeated copies of a short dsDNAcontaining a sequence or sequences that act as transcription factordecoys.

In another embodiment, the present invention provides for the use of thepolymers for covalent addition of targeting peptides, receptor bindingpeptides/protein domains and antibody fragments that may be used totarget the CODN/polymer complexes to a specific cell type; thus theagent can be made organ, tissue and/or cell-type specific.

In another embodiment, the present invention provides for usingpolyamides for targeting peptides and/or antibodies for specific stressand/or drug induced cellular receptors. In one embodiment, thepolyamides target the CODN/polymer complexes to ischemic, inflamed orcancerous tissues.

In another embodiment, the present invention provides for using linkerpeptides containing the sequence recognized by the TNF-alpha convertingenzyme (TACE) or another exopeptidase or endopeptidase in order to allowthe agent to deliver the CODN/polymer complex to the cell and thencleave off the targeting peptide.

In another embodiment, the present invention provides for using thepolyamides to deliver intact genes (transgenes), plasmids, RNAi, siRNA,morpholinos or other kinds of RNA, proteins and polynucleotides. In oneembodiment, the genes incorporate tissue-specific promoters,controllable promoters, promoters that may be silenced by specificCODN/polymer combinations and may constitute two- and three-unit systemsfor gene expression, control and DNA transposition (i.e. insertion,excision and targeting of transgenes and other DNA molecules).

In another embodiment, the present invention provides for use of thepolyamides in vitro or in vivo, in isolated cells or intact animals inwhich specific blockade of transcription factors or delivery of DNA orother biological effector is desirable. In one embodiment, this includesuse as a research tool, including studies of specific genes and studiesto identify specific genes regulated by the transcription factorstargeted (relates to development of specific CODN/polymer complex andrelated gene marker mouse lines described below). For clinical use, thiswould include, but is not limited to delivery of transcription factordecoys (e.g. CODNs) that block transcription factors implicated indisease, response to surgery and/or trauma, developmental defects,aging, toxic exposure, etc.

In another embodiment, the present invention provides for transgenicmice expressing marker genes (lacZ and/or GFP variants) under thecontrol of promoter elements that are primarily controlled by specifictranscription factors. In one embodiment, the mice are providedseparately or as a kit including specific CODN/polymer complexes and thematching mouse, which serves to identify the cells in which the markeractivation (experimentally activated) is blocked by the CODN. In anotherembodiment, there are transgenic mice with marker genes that aretranscriptionally turned on, which can be specifically turned off usingCODN/polymer cokplexes.

In another embodiment, the present invention provides for bi-transgenic(or multiple transgenic) systems designed to utilize the CODN/polymercokplexes to regulate gene expression (up, down, on or off) or tomediate gene transposition (insertion, excision or moving in thegenome).

In another embodiment, the present invention provides for polymersdesigned for variable release/biodegradation; some may be designed,selected for quick degradation/release of CODN, others for longhalf-life (the CODN may be active whether or not it is released by thepolymers, so we should safeguard the concept that long-lasting bindingof the DNA by polymers may be a way to prolong activity).

In another embodiment, the present invention provides for the deliveryof one or more imaging agents for real-time and still imaging within acell or tissue.

In another embodiment, the present invention provides for usingpolyamides for delivery of transcription factor decoys to blocksignaling and gene expression associated with pathogenesis.

In another embodiment, the present invention provides for usingpolyamides for delivery of linear duplications or chains of these decoys(i.e., concatemers), such that each strand contains a number of decoytranscription factor binding sites including more than 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, or more. A concatemer is a segment composed ofrepeated sequences linked end to end. In one embodiment, the concatemermay form a circular nucleotide.

In another embodiment, the present invention provides for usingpolyamides for delivery of decoys having for multiple transcriptionfactors into one of these strands, such that it can affect blockade of2, 3, 4, 5, 6, or more transcription factors simultaneously in a cell.In another embodiment, the present invention provides for usingpolyamides for delivery of these strands, or the strands contained in aplasmid or other DNA vector (can include phage, viral or other DNA) tobind to the polymers to deliver the strands to the cytoplasm of thecell, to effect transcription factor blockade.

Oligonucleotide Decoys

In one embodiment, the present invention relates to the use ofoligonucleotide decoys and/or concatemers for the production of amedicament for the therapy of NF-κB-dependent diseases. The presentinvention also relates to the prevention and treatment of variousdiseases associated with NF-κB which is known to be a regulatory factorin the transcription of cytokines and adhesion factors. Moreparticularly, the invention relates to a novel form of NF-κB decoy, acomposition comprising the decoy for the therapy and prophylaxis ofNF-κB-associated diseases, and a method for the therapy and prophylaxis.

In another embodiment, the present invention provides for using deliveryagent for NF-kB-specific CODN delivery in the treatment of myocardialischemia/reperfusion and myocardial infarction, heart failure andhypertrophy, cardioprotection, stroke, neuroprotection, sepsis,arthritis, asthma, heritable inflammatory disorders, cancer, heritableimmune dysfunctions, inflammatory processes, whether caused by diseaseor injury or infection, oxidative stress to any organ whether caused bydisease, surgery or injury. In another embodiment, the present inventionprovides for using delivery agents for delivery of CODN's to delineatein animal models, specific situations in which NF-kB or othertranscription factors contribute to injury, dysfunction, morbidity ormortality, determine whether blockade is beneficial in animals and thentranslating this to the clinic.

An important step in many inflammatory processes is the translocation ofthe protein “nuclear factor kappa B” to the nuclear compartment of thecell; in brief, translocation of NF-κB, into the cell nucleus and thestimulation of the expression of the genes caused thereby, whoseproducts are responsible for inflammatory reactions. For example, inasthma the nonbeneficial, excessive (non self-limiting) production ofthese proteins is responsible for the intensification and maintenance ofthe inflammatory process and the unpleasant to life-threatening symptomsof this disease associated therewith. Because the long-term treatmentwith glucocorticoids corresponding to the present state of the art isaffected by some severe and often debilitating disadvantages, NF-κB isseen as a compelling target for the development of new anti-inflammatoryactive compounds against asthma.

The oligonucleotide decoy substances utilizable according to theinvention are inhibitors which selectively inhibit nuclear factor kappaB (NF-κB)-mediated pathophysiological processes. NF-κB-mediatedprocesses occur in inflammatory diseases, immunological disorders,septic shock, transplant rejection, radiation damage, reperfusioninjuries after ischemia, hypoxia, asthma, cardiomyopathy, cardiachypertrophy, heart failure, muscle wasting, thromboses or in complex,chronic inflammatory disorders such as arteriosclerosis.

Nuclear factor kappa B (NF-κB) is a dimeric protein complex occurring inmany tissue cells and in particular in blood cells. NF-κB takes on aparticular role in the control of the expression of genes which have anNF-κB binding sequence (5′-GGGPuNNPyPyCC-3′) in their promoter sequence.To this extent, NF-κB is a transcription factor. The physiologicalactivity of NF-κB in the control of gene expression, however, is subjectto a regulation principle, in which NF-κB is released from a complexwith proteins of the IκB class in order to be translocated as atranscription factor to the cell nucleus resulting in gene activation.The regulation principle for the release of active NF-κB from a complexwith the protein IκB is still not known in detail.

Likewise, it is not known how the formation of homodimeric andheterodimeric NF-κB protein complexes takes place. NF-κB acts on geneactivation as a dimeric transcription factor. The dimerization can takeplace under the structurally related transcription factors Rel A, Rel B,c-Rel, p50 or p52, which form a family of transcription factor proteins.

The formation of different transcription factor dimmers provides somedegree of selectivity for different NF-kB DNA binding sites in vivo indifferent promoters. For instance, p65/p50 and p50/p50 as well asp65/p65 dimers are known to bind different specific NF-kB binding sites,that vary by only a few bases, with different affinities. Thesedifferent NF-kB dimmers may have distinct effects, such as differentlevels of gene activation (P65/p50 vs. p65/p65) and gene repression(p50/p450). This is thought to provide a basis for variation of NF-kBregulatory effects upon different genes, but is not yet understood indetail.

This selectivity of NF-kB dimmers could be taken advantage of in oneembodiment of the invention by varying the specific decoy sequence inthe CODN or by using specific combinations of variant decoys tospecifically blockade specific NF-kB dimmers. Furthermore, this NF-kBvariant dimmers may be precisely and selectively blocked relative to oneanother by controlling the relative number of the specific variant decoysites in a CODN sequence. Future investigations may reveal utility ofthis capability in specific diseases.

A crucial feature of NF-κB compared to other transcription factors isthat NF-κB is a primary transcription factor. Primary transcriptionfactors are already present in the cell in inactive (usuallycomplex-bound) form and are released after an appropriate stimulus inorder to be able to display their action very rapidly. Primarytranscription factors are not first formed by the activation of theassociated gene and subsequent transcription and translation.

This property of NF-κB, the formation of homodimeric or heterodimericRel proteins and the formation of an inactive protein complex with anIκB protein, offer very different points of attack for pharmacologicallyactive substances than the points of attack of the de novo biosynthesisof transcription factors. For the sake of completeness, it may bementioned that the genes for the formation of NF-κB (genes of the Relfamily) and the genes for the formation of the IκB proteins (gene familycomprising the genes for IκB-α, IκB-beta, p105/IκB-gamma,p100/IκB-delta, IκB-epsilon and others) for their part are of coursealso subject to regulation, by NF-kB itself, among other factors, whichcan be points of attack for pharmaceutically active substances. Thus itis known that the expression of the constitutively formed IκB proteinsp105 and p100 is increased by stimuli which also activate NF-κB, such astumour necrosis factor-α (TNF-α) or phorbol myristate acetate (PMA).However, blocking expression of NF-kB constituent and regulatory factorswill not acutely affect NF-kB activation since previously synthesizedNF-kB remains for some time. Thus, the novel aspect of the inventioncomes into play, allowing for immediate blockade of NF-kB activation bytitration (binding) of existing NF-kB molecules.

A regulation mechanism is described in the literature in which it isshown that the overexpression of IκB binds active NF-κB and thusinactivates it. This also applies if the NF-κB has already entered intoa complex with the DNA (P. A. Baeuerle, T. Henkel, Annu. Rev. Immunol.12, 141-179, 1994). From this it can be concluded that there are anumber of specific points of attack in the biochemical function of NF-κBand IκB proteins which should make it possible to inhibit anundesirable, pathophysiological, NF-κB-dependent gene activationselectively.

A chemical compound which selectively inhibits the function of NF-κB orthe function of IκB proteins or IκB genes to an increased extent shouldbe able to be used as a pharmaceutical for the suppression ofNF-κB-mediated disease processes.

Primarily, NF-κB can promote all pathophysiological processes in whichgenes are involved which have the NF-κB binding sequence in theirpromoter. Mainly, these are genes which play a crucial causal role inimmunological complications, in inflammatory diseases, autoimmunedisorders, septic shock, transplant rejection, cell death, cancer,asthma, thromboses or else alternatively in chronic inflammatorydiseases such as arteriosclerosis, arthritis, rheumatism and psoriasis.

NF-κB binding sequences are contained, for example, in the promoters ofreceptors of lymphoid cells (T-cell receptors), of MHCI and MHCII genes,of cell adhesion molecules (ELAM-1, VCAM-1, ICAM-1), of cytokines andgrowth factors (see also the following table). Furthermore, NF-κBbinding sequences are found in the promoters of acute phase proteins(angiotensinogen, complement factors and others).

A chronically increased or acutely overshooting activation of the genesmentioned leads to various pathophysiological processes and syndromes.

The rapid and overshooting production of cytokines and chronicmaintenance of pathological expression of the inflammatory reaction(TNFα, interleukin-2, interleukin-6, interleukin-8 and others) and ofthe adhesion molecules (ELAM-1, ICAM-1, VCAM-1) in leukocytes, inparticular in macrophages and also in endothelial cells andcardiomyocytes, is a causal feature of processes which often run a fatalcourse in the case of septic shock; or in the case of radiation damageand in the case of transplant rejection often leads to considerablecomplications. Inhibitors which prevent the NF-κB-mediated geneexpression intervene very early in some diseases in the expression ofpathophysiological changes and can therefore be a very effectivetherapeutic principle. An example is also NF-κB inhibitors for diseaseswhich are to be attributed to an overexpression of acute-phase proteins.An undesirable overexpression of acute-phase proteins can cause acomplex general reaction in which tissue damage of very different types,fever and local symptoms such as inflammation and necroses can occur.

Levels of specific serum proteins and circulating cell types are usuallychanged as is regulation of both the adaptive and innate immune systemfunction; all of these are known to be affected by NF-kB. NF-κB stronglyinduces, for example, the serum amyloid A precursor protein in the liverin the course of induction of acute-phase proteins.

For example, the NF-κB-mediated gene expression of theinterleukin-2-(II-2) gene can be inhibited.

Interleukin-2 is a cytokine, which plays a central role in variousinflammatory processes, inter alia, as a hematopoietic growth factor(Annu. Rev. Immunol. 1994, 12: 141-79). The promoter of theinterleukin-2 gene is NF-κB dependent. An inhibitor of NF-κB stimulationthus opens up the possibility of preventing overshooting of II-2production and thus of treating inflammatory processes.

In the case of other syndromes such as tissue damage after reperfusionor cirrhosis of the liver, inhibitors of NF-κB-mediated gene expressioncan likewise represent an important therapeutic advance. There isevidence that NF-κB-controlled genes are induced as a result ofoxidation reactions which lead to oxidative stress after reperfusion ofischemic tissue. In this way, an overexpression of cytokines and celladhesion molecules in the ischemic tissue causes excessive recruitmentof infiltrating alymphocytes. The recruited lymphocytes contributecausally to the tissue damage.

The involvement of NF-κB-controlled gene expression is evident in anumber of neurodegenerative disorders. In particular in the case ofnervous diseases in which the redox state of cells of the neuronaltissue is disturbed, a therapeutic benefit is ascribed to the selectiveinhibition of genes having an NF-κB binding sequence. A disturbed redoxstate of neuronal cells is assumed in the case of ainyotropic lateralsclerosis and in Down's syndrome.

It is therefore the general object of this invention to provides amethod of inhibiting or preventing cell death or apoptosis inischemic-reperfused myocardium using novel oligonucleotide decoys orCODNS.

The present invention provides a method for inhibiting cell death andapoptosis in ischemic-reperfused myocardium by administering to a mammalan effective amount of oligonucleotide and/or concatameric decoy, toreduce or prevent myocardial cell death in myocardial infarction.Furthermore, oligonucleotide decoys, either alone or conjugated to apolymeric vector or polyplex group, can be used to block apoptosis insituations of acute trauma, such as generalized trauma, globalischemia-reperfusion injury occurring as a consequence of hemorrhagicshock, or spinal cord injury, thereby preventing cell death in organssuch as the spinal cord.

In another embodiment, the present invention provides for the use forpolymers to deliver a concatemer to a cell. In another embodiment, thepresent invention provides for the use for polymers to deliver aconcatemerized double-stranded oligonucleotide molecules (CODN) fortranscription factor decoys. In one embodiment, the concatemers consistof a variable number of end-to-end repeated copies of a short (20-30 bp)dsDNA containing a sequence or sequences that act as transcriptionfactor decoys.

In another embodiment, the present invention provides for the use ofdelivery agents for covalent addition of targeting peptides, receptorbinding peptides/protein domains and antibody fragments that may be usedto target the CODN's to a specific cell type; thus the agent can be madeorgan, tissue and/or cell-type specific.

In another embodiment, the present invention provides for using polymersfor targeting peptides and/or antibodies for specific stress and/or druginduced cellular receptors. In one embodiment, the polymers target theCODN's to ischemic, inflamed or cancerous tissues.

In another embodiment, the present invention provides for using linkerpeptides containing the sequence recognized by the TNF-alpha convertingenzyme (TACE) or another exopeptidase or endopeptidase in order to allowthe agent to deliver the CODN's to the cell and then cleave off thetargeting peptide.

In another embodiment, the present invention provides for using thepolymers to deliver intact genes (transgenes), plasmids, RNAi, siRNA,morpholinos or other kinds of RNA, proteins and polynucleotides. In oneembodiment, the genes incorporate tissue-specific promoters,controllable promoters, promoters that may be silenced by specificCODN's and may constitute two- and three-unit systems for geneexpression, control and DNA transposition (i.e. insertion, excision andtargeting of transgenes and other DNA molecules).

In another embodiment, the present invention provides for use of thepolymers in vitro or it vivo, in isolated cells or intact animals inwhich specific blockade of transcription factors or delivery of DNA orother biological effector is desirable. In one embodiment, this includesuse as a research tool, including studies of specific genes and studiesto identify specific genes regulated by the transcription factorstargeted (relates to development of specific CODN and related genemarker mouse lines described below). For clinical use, this wouldinclude, but is not limited to delivery of transcription factor decoys(e.g. CODNs) that block transcription factors implicated in disease,response to surgery and/or trauma, developmental defects, aging, toxicexposure, etc.

In another embodiment, the present invention provides for transgenicmice expressing marker genes (lacZ and/or GFP variants) under thecontrol of promoter elements that are primarily controlled by specifictranscription factors. In one embodiment, the mice are providedseparately or as a kit including specific CODN/polymer complexes and thematching mouse, which serves to identify the cells in which the markeractivation (experimentally activated) is blocked by the CODN. In anotherembodiment, there are transgenic mice with marker genes that aretranscriptionally turned on, which can be specifically turned off usingCODN/polymer complexes.

In another embodiment, the present invention provides for bi-transgenic(or multiple transgenic) systems designed to utilize the CODN/polymercomplexes to regulate gene expression (up, down, on or off) or tomediate gene transposition (insertion, excision or moving in thegenome). In one embodiment, transgene A may express a gene of interestunder control of a promoter that is inducible by NF-kB or by a yeast orbacterial transcription factor (think tetR or Gal4). In one embodiment,the gene would be on after an NF-kB-inducing stimulus, or constitutivelyon in a tissue expressing the specific transcription factor (we aremaking mice for NF-kB activation; mice for gal4 and tetR already exist)and the gene could be turned off by simply providing the CODN/polymercomplexes for the specific transcription factor (CODN-OFF). In anotherembodiment, the animals are continuously delivered CODN/polymer complexand then the CODN/polymer complex is withdrawn to turn the gene on.Other versions could have the gene off, due to expression in the samecells of a transcriptional repressor (has been described for tet), andthe repression reversed by adding CODN/polymer complex, allowingexpression to turn on (CODN-ON).

Another embodiment provides for the delivery of transgenes that may beincorporated into the genome via retroviruses, transposons orretrotransposons. In one embodiment, the delivery is for long-term geneexpression or genetic engineering in vitro, in vivo, in isolated cellsor in whole animals or in the clinic. In another embodiment, germ cellsare targeted using compositions of the present invention to achieveheritable transgenic lines of animals without having to domicroinjection (optionally using a bi-transgenic system).

In another embodiment, the present invention provides for using deliveryagents for delivery of transcription factor decoys (including, but notlimited to NF-kB), to block signaling and gene expression associatedwith pathogenesis.

In another embodiment, the present invention provides for using polymersfor delivery of linear duplications or chains of these decoys (i.e.,concatemers), such that each strand contains a number of decoytranscription factor binding sites including more than 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30 or more. In another embodiment, the presentinvention provides for using polymers for delivery of decoys having formultiple transcription factors into one of these strands, such that itcan affect blockade of 2, 3, 4, 5, 6, or more transcription factorssimultaneously in a cell. In another embodiment, the present inventionprovides for using polymers for delivery of these strands, or thestrands contained in a plasmid or other DNA vector (can include phage,viral or other DNA) to bind to the polymers to deliver the strands tothe cytoplasm of the cell, to effect transcription factor blockade.

In an alternate embodiment, the decoy transcription factor binding siteson each strand may be separated via a spacer. A spacer element isgenerally a nucleic acid, that is to say, it comprises nucleic acidresidues. The nucleic acids may be naturally occurring or non-naturallyoccurring. A spacer may comprise two or more nucleic acids. It may, forexample, comprise three or more nucleic acids, for example, four ormore, for example, five or more, for example, up to ten nucleic acids ormore. The nucleic acids may be the same or different.

The decoys may be any transcription factors, including, but not limitedto, NF-kB, AP-1, ATF2, ATF3, SP1 and others. This is all based on thenovel concept, supported by data in our lab, that blocking key signalingmolecules simultaneously can have additive or even synergistictherapeutic effects, particularly when the molecules chosen are keysignaling hubs. In signaling, transcription factors participate byactivating or turning down gene expression. In another embodiment, thepresent invention provides for using CODN's for treatment of MI byblocking NF-kB using decoys to iNOS and Cox2. In another embodiment, thepresent invention provides for using delivery agents for delivery ofdecoys to metallothionein and heat shock protein 70.

We are using transcription factor decoys (including, but not limited tothe one for NF-kB, disclosed), to block signaling and gene expressionassociated with pathogenesis. The data that we have pertains to blockingNF-kB in the heart, which we have shown is efficacious in reducingmyocardial infarction. However, the concept that we wish to disclosetakes this several steps further. First, we are and will use linearduplications or chains of these decoys, such that each strand contains anumber of decoy transcription factor binding sites with more than 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or more copies linked tostabilize the decoy and increases efficacy.

In another embodiment, we incorporate decoys for multiple transcriptionfactors into one of these strands, such that the decoy can affectblockade of 2 or more transcription factors simultaneously in a cell. Inanother embodiment, we incorporate decoys for multiple transcriptionfactors into one of these strands, such that the decoy can affectblockade of 3 or more transcription factors simultaneously in a cell.Furthermore, the number of sites specific for different factors can beprecisely determined so as to precisely control the extent of blockade(there can be simultaneous differential titration of blockade formultiple factors). This is important, since in many diseases andbiological processes, the relative amount of activation of multiplesignaling pathways controls biological output and disease progression.

Third, we use these strands, or the strands contained in a plasmid orother DNA vector (including, without limitation, phage, viral or otherDNA, and these can be concatemers engineered by recombinant DNAtechniques) to bind to the polyplexes to deliver the strands to thecytoplasm of the cell, to effect transcription factor blockade.

Preferably, the oligonucleotide decoy inhibits one or more transcriptionfactor. More preferably, the oligonucleotide decoy inhibits NF-kB inaddition to one or more transcription factor selected from the groupconsisting of AP-1, ATF2, ATF3, SP1 and related factors. By blocking keysignaling molecules simultaneously has an additive or even synergistictherapeutic effect, particularly when the molecules chosen are keysignaling hubs.

In another embodiment, the domains can include decoys to specificpromoters, including but not limited to one or more of NF-kB, iNOS,Cox2, metallothionein and heat shock protein 70.

The use of long chains (concatemers) of the binding sites to NF-kB andrelated genes, the decoy is able to bind much more NF-kB or relatedmolecules per molecule and to effect binding to the polymers.Furthermore, one can deliver much more of the decoy with relatively lessof the polymer, which may have specific advantages, including reducedside effects of the polymer.

Generally, the term “oligonucleotide,” refers to a short length ofsingle-stranded polynucleotide chain. Oligonucleotides are typicallyless than 100 residues long (e.g., between 15 and 50), however, as usedherein, the term is also intended to encompass longer polynucleotidechains. Oligonucleotides are often referred to by their length. Forexample a 22 residue oligonucleotide is referred to as a “22-mer”.Oligonucleotides can form secondary and tertiary structures byself-hybridizing or by hybridizing to other polynucleotides. Suchstructures can include, but are not limited to, duplexes, hairpins,cruciforms, bends, and triplexes.

As used herein, the term “transcription factor” refers to proteins thatinteract with one another and RNA polymerase enzyme to modulatetranscription. Transcription factors target genes by recognizingspecific DNA regulatory sequences (e.g., promoters and enhancers) orother transcription factors. Transcription factors are often referred toas “trans-factors” that interact with “cis-elements” (e.g., enhancers)because they are typically produced from genes located distantly (trans)from their sites of regulation (cis). Some transcription factors arebiologically active only when bound to another copy of itself (i.e.,homodimers linked through “homodimerization domains”) or to othertranscription factors (i.e., heterodimers linked through“heterodimerization domains”. For most transcription factors, specificand distinct regions of the protein mediate DNA binding (i.e., “tDNAbinding domains”) and transcriptional activation (i.e., “activationdomains”).

A “biologically active compound” is a compound having the potential toreact with biological components. More particularly, biologically activecompounds utilized in this specification are designed to change thenatural processes associated with a living cell. For purposes of thisspecification, a cellular natural process is a process that isassociated with a cell before delivery of a biologically activecompound. Biologically active compounds may be selected from the groupcomprising: pharmaceuticals, proteins, peptides, polypeptides, hormones,cytokines, antigens, viruses, oligonucleotides, nucleic acids, andsynthetic polymers such as polypyroles could also be delivered.

As used herein, the term “transfection” means the process of deliveringa polynucleotide to a cell has been commonly termed transfection or theprocess of transfecting and also it has been termed transformation. Theterm transfecting as used herein refers to the introduction of apolynucleotide or other biologically active compound into cells. Thepolynucleotide may be used for research purposes or to produce a changein a cell that can be therapeutic. The delivery of a polynucleotide fortherapeutic purposes is commonly called gene therapy. The delivery of apolynucleotide can lead to modification of the genetic material presentin the target cell. The term stable transfection or stably transfectedgenerally refers to the introduction and integration of an exogenouspolynucleotide into the genome of the transfected cell. The term stabletransfectant refers to a cell which has stably integrated thepolynucleotide into the genomic DNA. Stable transfection can also beobtained by using episomal vectors that are replicated during theeukaryotic cell division (e.g., plasmid DNA vectors containing apapilloma virus origin of replication, artificial chromosomes). The termtransient transfection or transiently transfected refers to theintroduction of a polynucleotide into a cell where the polynucleotidedoes not integrate into the genome of the transfected cell. If thepolynucleotide contains an expressible gene, then the expressioncassette is subject to the regulatory controls that govern theexpression of endogenous genes in the chromosomes. The term transienttransfectant refers to a cell which has taken up a polynucleotide buthas not integrated the polynucleotide into its genomic DNA.

The term “transfection agent” or “transfection reagent” or “deliveryvehicle”, is a compound or compounds that bind(s) to or complex(es) witholigonucleotides and polynucleotides, and enhances their entry intocells. Examples of transfection reagents include, but are not limitedto, cationic liposomes and lipids, polyamines, calcium phosphateprecipitates, histone proteins, polyethylenimine, polylysine, andpolyampholyte complexes. Other reagents include cationic proteins likehistones and protamines, or synthetic polymers like polylysine,polyarginine, polyornithine, DEAE dextran, polybrene, andpolyethylenimine may be effective intracellular in vitro deliveryagents. Typically, the transfection reagent has a component with a netpositive charge that binds to the oligonucleotide's or polynucleotide'snegative charge. For delivery in vivo, complexes made withsub-neutralizing amounts of cationic transfection agent may bepreferred. Non-viral vectors is include protein and polymer complexes(polyplexes), lipids and liposomes (lipoplexes), combinations ofpolymers and lipids (lipopolyplexes), and multilayered and rechargedparticles. Transfection agents may also condense nucleic acids.

The term “polynucleotide”, or “nucleic acid” or “polynucleic acid”, is aterm of art that refers to a polymer containing at least twonucleotides. Nucleotides are the monomeric units of polynucleotidepolymers. Polynucleotides with less than 120 monomeric units are oftencalled oligonucleotides. Natural nucleic acids have a deoxyribose- orribose-phosphate backbone. An artificial or synthetic polynucleotide isany polynucleotide that is polymerized in vitro or in a cell free systemand contains the same or similar bases but may contain a backbone of atype other than the natural ribose-phosphate backbone. These backbonesinclude: PNAs (peptide nucleic acids), phosphorothioates,phosphorodiamidates, morpholinos, and other variants of the phosphatebackbone of native nucleic acids. Bases include purines and pyrimidines,which further include the natural compounds adenine, thymine, guanine,cytosine, uracil, inosine, and natural analogs. Synthetic derivatives ofpurines and pyrimidines include, but are not limited to, modificationswhich place new reactive groups such as, but not limited to, amines,alcohols, thiols, carboxylates, and alkylhalides. The term baseencompasses any of the known base analogs of DNA and RNA. The termpolynucleotide includes deoxyribonucleic acid (DNA) and ribonucleic acid(RNA) and combinations of DNA, RNA and other natural and syntheticnucleotides.

As used herein, the terms “decoy” and “transcription factor decoy” referto molecules that bind to or interact with transcription factors andprevent their binding to native enhancer and promoter sequences. Decoysinclude nucleic acid sequences, including, but not limited to,oligonucleotides that correspond to (i.e., are identical to oressentially identical to) the native promoter or enhancer. Sucholigonucleotides include, but are not limited to, single strandedpalindromic oligonucleotides comprising one or more repeats of thepromoter or enhancer sequence, sense and antisense oligonucleotidescomprising one or more repeats of the promoter or enhancer sequence,oligonucleotides or other artificial gene products (e.g., mRNAs) thatform hairpin structures such that a duplex binding site for thetranscription factor is generated, and one or more oligonucleotides thatform a cruciform structure such that one or more binding sites for thetranscription factor are generated.

As used herein, the term “duplex,” in reference to oligonucleotides,refers to regions that are double stranded through hybridization ofcomplementary base pairs. The term “hairpin” refers to double-strandednucleic acid structures formed by base-pairing between regions of thesame strand of a nucleic acid molecule. The regions are arrangedinversely and can be adjacent or separated by noncomplementary sequence(i.e., thus forming a loop structure or “stem-loop”). The term“cruciform” refers to structures formed in double-stranded nucleic acidsby inverted repeats separated by a short sequence. Cruciform structurescan be generated through the hybridization of two or more hairpinstructures where the hairpin duplex and loop comprise the short sequenceseparating the inverted repeats. Cruciform structures can comprise oneor more nucleic acid molecules.

In an alternate embodiment, the polynucleotide decoys of the inventioncomprise an internal oligonucleotide (I) having a length of X bases,where X is a number from about 10 to about 40, preferably 12 to 25, mostpreferably 14 to 20. The size of the I segments is bounded on the lowerend by their ability to maintain the relative binding affinity of thelarger segments to, for example, transcription factors. The size of theI segments is bounded on the upper end by their ability to remainrelatively insensitive to endonucleases. Thus, the length limits of theI segment of a decoy can be determined empirically by one of skill inthe art.

In an alternate embodiment, the polynucleotide decoys of the inventionfurther comprise cap or spacer oligonucleotides, having a length of fromabout 3 to about 24 bases, preferably 4 to 18 base most preferably 6 to12 bases. Each of the cap or spacer oligonucleotides is comprised ofbases that are unable to bind to any other base within the same capoligonucleotide. Preferably each of the cap or spacer oligonucleotidesconsists of a single variety of nucleotide comprising a base selectedfrom the group consisting of adenine, cytosine, thymidine, and modifiednucleotides thereof.

In an alternate embodiment, the polynucleotide decoys of the inventioncomprise a formula comprising: (a) an internal oligonucleotide (I)having a length of X bases, where X is a number from about 14 to about40; (b) a second complementary oligonucleotide (C₂) having a length of Zbases, where Z is a number greater than

Preferably, the domains (I) are covalently linked to the 5′ end of theP1, the 3′ end of the P1 is covalently linked to the 5′ end of next I,the 3′ end of the I is covalently linked to the 5′ end of the P2, andthe 3′ end of the P2 is covalently linked to the 5′ end of the next (I).In a specific embodiment, the polynucleotide comprises at least 10, 20,30, 40, 50, 60, 70, 80, 90, 100, or more domains (I) linked togetherwith spacers. In one embodiment, the polynucleotide comprises at leasttwo different domains (I) throughout the molecule.

The invention also provides for a purified decoy probe comprising afirst nucleotide base recognition sequence region, wherein the firstregion binds to a transcription factor, and an optionally present secondnucleotide base recognition sequence region, provided that if the firstregion is nucleic acid and the second region is present, then the secondregion is either directly joined to the 5′ end of the first region isjoined to the 3′ end or 5′ end of the first region by a non-nucleotidelinker, wherein the optionally present second region is present if thefirst region can be used to produce a functional double-strandedpromoter sequence using a complementary oligonucleotide, furtherprovided that if the first region is nucleic acid which can be used toproduce the functional double-stranded promoter sequence using thecomplementary oligonucleotide, then the decoy probe does not have anucleic acid sequence greater than about 10 nucleotides in length joineddirectly to the 3′ end of the first region and the decoy probe does nothave a terminal 3′ OH group available to accept a nucleosidetriphosphate in a polymerization reaction.

The diseases in which the therapeutic/prophylactic composition of theinvention is indicated are NF-κB-associated diseases, that is to saydiseases caused by the unwanted activation of genes under control of thetranscriptional regulatory factor NF-κB, and among such diseases can bereckoned ischemic diseases, hypoxic conditions, ischemic andpharmacologic preconditioning, surgical trauma, cardiac hypertrophy,cardiomyopathy, heart failure, inflammatory diseases, autoimmunediseases, cancer metastasis and invasion, and cachexia. The ischemicdisease includes ischemic diseases of organs (e.g. ischemic heartdiseases such as myocardial infarction, acute heart failure, chronicheart failure, etc., ischemic brain diseases such as cerebralinfarction, and ischemic lung diseases such as pulmonary infarction),aggravation of the prognosis of organ transplantation or organ surgery(e.g. aggravation of the prognosis of heart transplantation, cardiacsurgery, kidney transplantation, renal surgery, liver transplantation,hepatic surgery, bone marrow transplantation, skin grafting, cornealtransplantation, and lung transplantation), reperfusion disorders, andpost-PTCA restenosis. The inflammatory disease mentioned above includesvarious inflammatory diseases such as nephritis, hepatitis, arthritis,etc., acute renal failure, chronic renal failure, and arteriosclerosis,among other diseases. The autoimmune disease mentioned above includesbut is not limited to rheumatism, multiple sclerosis, and Hashimoto'sthyroiditis. Particularly the pharmaceutical composition containing theNF-κB decoy according to the present invention as an active ingredientis very suited for the therapy and prophylaxis of reperfusion disordersin ischemic diseases, aggravation of the prognosis of organtransplantation or organ surgery, post-PTCA restenosis, cancermetastasis and invasion, and cachexia such as weight loss following theonset of a cancer.

The NF-κB decoy that can be used in the present invention may be anycompound that specifically antagonizes the NF-κB binding site of thechromosomes and includes but is not limited to nucleic acids and theiranalogs. As preferred examples of the NF-κB decoy, the present inventionmay utilize NF-kB decoy comprising one or more copies ofoligonucleotides CCTTGAAGGGATTTCCCTCC and GGAACTTCCCTAAAGGGAGG,preferably, the NF-kB decoy are described as oligonucleotides containingthe nucleotide sequence of GGGATTTCCC. Preferably, the NF-kB decoyoligonucleotide is a double-stranded 22 bp oligonucleotide(5′-AGTTGAGGGGACTTTCCCAGGC-3′) (Promega).

The oligonucleotides may be DNAs or RNAs, and may contain modifiednucleotides and/or pseudonucleotides. Furthermore, thoseoligonucleotides, variants thereof, or compounds containing any of themmay be single-stranded or double-stranded and linear or cyclic. Thevariants are those involving mutations such as substitution, additionand/or deletion of any part of the above-mentioned sequence, and meannucleic acids specifically antagonizing the binding site of chromosometo which NF-κB is conjugated. The more preferred NF-κB decoy includesdouble-stranded oligonucleotides each containing one or a plurality ofthe above nucleotide sequence and variants thereof. The oligonucleotidewhich can be used in the present invention includes oligonucleotidesmodified so as to be less susceptible to biodegradation, such as thoseoligonucleotides containing the thiophosphoric diester bond availableupon substitution of sulfur for the oxygen of the phosphoric diestermoiety (S-oligo) and those oligonucleotides available upon substitutionof a methyl phosphate group carrying no electric charge for thephosphoric diester moiety.

Regarding to a technology for producing the NF-κB decoy for use in thepresent invention, the conventional chemical, biochemical, or biological(including recombinant DNA) methods for synthesis can be utilized. Whena nucleic acid, for instance, is to be used as the NF-κB decoy, themethods for nucleic acid synthesis which are commonly used in geneticengineering can be employed. For example, the objective decoyoligonucleotide can be directly synthesized on a DNA synthesizer. Or anucleic acid or its fragments, each synthesized beforehand, can beamplified by PCR or using a cloning vector or the like. Furthermore, thedesired nucleic acid can be obtained by such procedures as cleavage withrestriction enzymes or the like and/or ligation by means of DNA ligaseor the like. In order to obtain a decoy nucleotide which is more stablewithin cells, the base, sugar or/and phosphoric acid moieties of thenucleic acid may be alkylated, acylated, or otherwise chemicallymodified, or designed to have a closed circular looped end (i.e. like adumbbell decoy).

The pharmaceutical composition containing the NF-κB decoy as an activeingredient according to the present invention is not limited in formonly if the active ingredient may be taken up by the cells in theaffected site or the cells of the target tissue. Thus, the NF-κB decoy,either alone or in admixture with the common pharmaceutical carrier, canbe administered orally, parenterally, topically or externally. Thepharmaceutical composition may be provided in liquid dosage forms suchas solutions, suspensions, syrups, liposomes, lotions, etc. or in soliddosage forms such as tablets, granules, powders, and capsules. Wherenecessary, those pharmaceutical compositions may be supplemented withvarious vehicles, excipients, stabilizers, lubricants, and/or otherconventional pharmaceutical additives, such as lactose, citric acid,tartaric acid, stearic acid, magnesium stearate, terra alba, sucrose,corn starch, talc, gelatin, agar, pectin, peanut oil, olive oil, caccaobutter, ethylene glycol, and so on.

Particularly when a nucleic acid or a modification product thereof isused as the NF-κB decoy, the preferred dosage form includes those whichare generally used in gene therapy, such as liposomes inclusive ofmembrane fusion liposomes utilizing Sendai virus and the like andliposomes utilizing endocytosis, preparations containing cationic lipidssuch as Lipofectamine (Life Tech Oriental) or virosomes utilizing aretrovirus vector, adenovirus vector, or the like. Particularlypreferred are membrane fusion liposomes.

The structure of such a liposomal preparation may be any of a largeunilamellar vesicle (LUV), a multi-lamellar vesicle (MLV), and a smallunilamellar vesicle (SUV). The approximate size of vesicles may rangefrom 200 to 1000 nm for LUV, from 400 to 3500 nm for MLV, and from 20 to50 nm for SUV but in the case of a membrane fusion liposomal preparationusing Sendai virus, for instance, MLV with a vesicular system of200-1000 nm in diameter is preferably employed.

There is no limitation on the technology for liposome production only ifthe decoy can be successfully entrapped in vesicles. Thus, suchliposomes can be manufactured by the conventional techniques such as thereversed phase evaporation method (Szoka, F., et al: Biochim. Biophys.Acta, Vol. 601 559 (1980)), ether injection method (Deamer, D. W.: Ann.N.Y. Acad. Sci., Vol. 308 250 (1978)), and surfactant method (Brunner,J., et al: Biochim. Biophys. Acta, Vol. 455 322 (1976)), to name but afew examples.

The lipid that can be used for constructing a liposomal structureincludes phospholipids, cholesterol and its derivatives, andnitrogen-containing lipids but phospholipids are generally preferred.The phospholipid that can be used includes naturally-occurringphospholipids such as phosphatidylcholine, phosphatidylserine,phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine,phosphatidic acid, cardiolipin, sphingomyelin, egg yolk lecithin,soybean lecithin, lysolecithin, etc., the corresponding phospholipidshydrogenated by the conventional method, and synthetic phospholipidssuch as dicetyl phosphate, distearoylphosphatidylcholine,dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine,dipalmitoylphosphatidylserine, eleostearoylphosphatidylcholine,eleostearoylphosphatidylethanolamine, eleostearoylphosphatidylserine,and so on.

The lipids inclusive of phospholipids can be used each alone or in asuitable combination. By using a lipid containing a positively-chargedatomic group such as ethanolamine or choline within the molecule, thebinding rate of an electrically negative decoy nucleotide can beenhanced. In addition to the principal phospholipid, various compoundssuch as cholesterol and its derivatives, stearylamine, -tocopherol,etc., which are known as liposome additives, can be added in themanufacture of liposomes.

To the resulting liposomes can be added a membrane fusion promoter suchas Sendai virus, inactivated Sendai virus, a membrane fusion promotingprotein purified from Sendai virus, polyethylene glycol, or the like canbe added for assisting in the intracellular uptake by the cells at theaffected site or of the target tissue.

There is no limitation on the decoy content of the pharmaceuticalcomposition containing the decoy as an active ingredient only if thedecoy is contained in amounts effective to control NF-κB-associateddiseases. Thus, the decoy content can be liberally selected according tothe disease to be controlled, the target site, dosage form, and dosageschedule.

The pharmaceutical composition containing the decoy as an activeingredient as provided in the above manner can be administered byvarious methods according to the type of disease and the kind of decoycontained. Taking ischemic diseases, inflammatory diseases, autoimmunediseases, cancer metastasis or invasion, and cachexia as examples, thecomposition can be infused intravascularly, applied directly to theaffected area, injected into the lesion, or administered into theregional blood vessel, tissue, or organ in the affected region. As afurther specific example, when PTCA is performed for infarction of anorgan, the pharmaceutical composition can be administered into the localblood vessel concurrently with the operation or pre- andpostoperatively. For organ transplantation, the graft material can bepreviously treated with the composition of the invention. Furthermore,in the treatment of osteoarthritis or rheumatism, the composition can bedirectly injected into the joint.

The dosage of the decoy is selected with reference to the patient's ageand other factors, type of disease, the kind of decoy used, etc. but forintravascular, intramuscular, or intraarticular administration, forinstance, a unit dose of 10-10,000 nmoles can generally be administeredonce to a few times daily.

As used herein, the term “procedural vascular trauma” includes theeffects of surgical/mechanical interventions into mammalian vasculature,but does not include vascular trauma due to the organic vascularpathologies listed hereinabove.

Thus, procedural vascular traumas within the scope of the presenttreatment method include (1) organ transplantation, such as heart,kidney, liver and the like, e.g., involving vessel anastomosis; (2)vascular surgery, such as coronary bypass surgery, biopsy, heart valvereplacement, atheroectomy, thrombectomy, and the like; (3) transcathetervascular therapies (TVT) including angioplasty, e.g., laser angioplastyand PTCA procedures discussed hereinbelow, employing balloon catheters,and indwelling catheters; (4) vascular grafting using natural orsynthetic materials, such as in saphenous vein coronary bypass grafts,dacron and venous grafts used for peripheral arterial reconstruction,etc.; (5) placement of a mechanical shunt, such as a PIFE hemodialysisshunt used for arteriovenous communications; and (6) placement of anintravascular stent, which may be metallic, plastic or a biodegradablepolymer. See U.S. patent application Ser. No. 08/389,712, filed Feb. 15,1995, which is incorporated by reference herein. For a generaldiscussion of implantable devices and biomaterials from which they canbe formed, see H. Kambic et al., “Biomatedals in Artificial Organs”,Chem. Eng. News. 30 (Apr. 14, 1986), the disclosure of which isincorporated by reference herein.

The present invention generally relates to coronary heart attacks andcardiovascular surgery, and other surgeries with cardiovascularcomplications. More particularly, the invention is related to the use ofoligonucleotide decoy as a protective agent during cardiac andneuronal/brain surgery and during the ischemia/reperfusion phases ofacute myocardial infarction (coronary heart attack) or stroke. Alsoincludes instances of peripheral ischemia and hypoxia that occurs as aresult of disease, trauma or as a complication of other procedures.

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein. The NFkB associatedpolynucleotides and polypeptides are sometimes referred to herein as“NFkB modulatory” polynucleotides and polypeptides. Likewise, allreferences to “NFkB associated polynucleotides and polypeptides” shallbe construed to apply to “NFkB modulatory polynucleotides andpolypeptides”.

The invention provides the polynucleotide and polypeptide sequences ofgenes that are believed to be associated with the NF-kB pathway. Asreferenced herein, members of the NFkB family are transcription factorsthat are critical regulators of inflammatory and stress responses. Thus,the polynucleotide and polypeptides of the present invention may also berepresent critical regulators of inflammatory and stress responses.

In the present invention, “isolated” refers to material removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring), and thus is altered “by the hand of man” from its naturalstate. For example, an isolated polynucleotide could be part of a vectoror a composition of matter, or could be contained within a cell, andstill be “isolated” because that vector, composition of matter, orparticular cell is not the original environment of the polynucleotide.The term “isolated” does not refer to genomic or cDNA libraries, wholecell total or mRNA preparations, genomic DNA preparations (includingthose separated by electrophoresis and transferred onto blots), shearedwhole cell genomic DNA preparations or other compositions where the artdemonstrates no distinguishing features of the polynucleotide/sequencesof the present invention.

In specific embodiments, the polynucleotides of the invention are atleast 15, at least 30, at least 50, at least 100, at least 125, at least500, or at least 1000 continuous nucleotides but are less than or equalto 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb,2.0 kb, or 1 kb, in length.

In a alternate embodiment, polynucleotides of the invention comprise aportion of the coding sequences, as disclosed herein, but do notcomprise all or a portion of any intron. In another embodiment, thepolynucleotides further comprise coding sequences coding sequences of agenomic flanking gene (i.e., 5′ or 3′ to the gene of interest in thegenome). In other embodiments, the polynucleotides of the invention donot contain the coding sequence of more than 1000, 500, 250, 100, 50,25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373, preferably a Model 3700, from AppliedBiosystems, Inc.), and all amino acid sequences of polypeptides encodedby DNA molecules determined herein were predicted by translation of aDNA sequence determined above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded thesequenced DNA molecule, beginning at the point of such an insertion ordeletion. Such point mutations can also change the DNA binding affinityof transcription factors and thus are worthy of consideration in thecurrent invention.

Also contemplated are nucleic acid molecules that hybridize to thepolynucleotides of the present invention at lower stringencyhybridization conditions. Changes in the stringency of hybridization andsignal detection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37 degree C. ina solution comprising 6.times.SSPE (20.times.SSPE=3M NaCl; 0.2M NaH2PO4;0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon spermblocking DNA; followed by washes at 50 degree C. with 1.times.SSPE, 0.1%SDS. In addition, to achieve even lower stringency, washes performedfollowing stringent hybridization can be done at higher saltconcentrations (e.g., 5.times.SSC).

Of course, a polynucleotide which hybridizes only to polyA+ sequences(such as any 3′ terminal polyA+ tract of a cDNA shown in the sequencelisting), or to a complementary stretch of T (or U) residues, would notbe included in the definition of “polynucleotide,” since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone generated using oligo dT as a primer).

The polynucleotide of the present invention can be composed of anypolyribonucleotide or polydeoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotide can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide may also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

Methods for introducing the compositions, complexes, nucleic acidmolecules and/or vectors of the invention into cells, tissues, organs ororganisms as described herein will be familiar to those of ordinaryskill in the art. For instance, the compositions, nucleic acid moleculesand/or vectors of the invention may be introduced into cells, tissues,organs or organisms using well known techniques of infection,transduction, transfection, and transformation. The compositions,nucleic acid molecules and/or vectors of the invention may be introducedalone or in conjunction with other compositions, nucleic acid moleculesand/or vectors. Alternatively, the compositions, nucleic acid moleculesand/or vectors of the invention may be introduced into cells, tissues,organs or organisms as a precipitate, such as a calcium phosphateprecipitate, or in a complex with a lipid. Electroporation also may beused to introduce the nucleic acid molecules and/or vectors of theinvention into a host. Likewise, such molecules may be introduced intochemically competent cells such as E. coli.

The polypeptide of the present invention can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well-described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result frompost-translation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-inking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells. The term “organism” as referred to herein is meantto encompass any organism referenced herein, though preferably toeukaryotic organisms, more preferably to mammals, including rats andmice, and most preferably to humans.

As used herein the terms “modulate” or “modulates” refer to an increaseor decrease in the amount, quality or effect of a particular activity,DNA, RNA, or protein. The definition of “modulate” or “modulates” asused herein is meant to encompass agonists and/or antagonists of aparticular activity, DNA, RNA, or protein.

Specifically, the invention provides methods for using thepolynucleotides and polypeptides of the invention to identify orthologs,homologs, paralogs, variants, and/or allelic variants of the invention.Also provided are methods of using the polynucleotides and polypeptidesof the invention to identify the entire coding region of the invention,non-coding regions of the invention, regulatory sequences of theinvention, and secreted, mature, pro-, prepro-, forms of the invention(as applicable).

In preferred embodiments, the invention provides methods for identifyingthe glycosylation and phosphorylation sites inherent in thepolynucleotides and polypeptides of the invention, and the subsequentalteration, deletion, and/or addition of the sites for a number ofdesirable characteristics which include, but are not limited to,augmentation of protein folding, inhibition of protein aggregation,regulation of intracellular trafficking to organelles, increasingresistance to proteolysis, modulation of protein antigenicity, andmediation of intercellular adhesion.

In further preferred embodiments, methods are provided for evolving thepolynucleotides and polypeptides of the present invention usingmolecular evolution techniques in an effort to create and identify novelvariants with desired structural, functional, and/or physicalcharacteristics.

While the NFkB-associated sequences are likely to compriserepresentatives from a number of protein families and classes (such asGPCRs, transcription factors, ion channels, proteases, nucleases,secreted proteins, nuclear hormone receptors, etc.), their biologicalactivity will likely not be exactly the same as NFkB (e.g., atranscription factor). Rather the NFkB associated polynucleotides andpolypeptides of the present invention are believed to represent eitherdirect, or indirect, participating members of the NFkB pathway.Therefore, it is expected that the NFkB associated polynucleotides andpolypeptides of the present invention, including agonists, antagonists,or fragments thereof, will be capable of providing at least some of thetherapeutic benefits afforded by modulators of NFkB, and potentiallyNFkB itself, upon administration to a patient in need of treatment. Thepresent invention also encompasses antagonists or agonists of thepolynucleotides and polypeptides, including fragments thereof, and theirpotential utility in modulating NFkB modulators, NF-kB-dependent genesand signaling pathways, and potentially NFkB itself.

Modulating the activity of the NFkB associated genes of the presentinvention may result in fewer toxicities than the drugs, therapies, orregimens presently known to regulate NF-kappaB itself (due to thespecificity inherent in specific embodiments, e.g., concatemers). SuchNF-kappaB inhibitors include the following, non-limiting examples: NFkBdecoy oligonucleotide-HVJ liposomes complex (Dainippon); genetherapy-based implantation of the I kappa B gene into donor organs exvivo (Novartis; EP699977); drugs designed to block IkappaBalpha-directedubiquitination enzymes resulting in more specific suppression of NF-κBactivation (Aventis); declopramide (OXIGENE; CAS Registry Number:891-60-1); IPL-550260 (Inflazyme); IPL-512602 (Inflazyme); KP-392(Kinetek); R-flurbiprofen (Encore Pharmaceuticals; E-7869, MPC-7869;(1,1′-Biphenyl)-4-acetic acid, 2-fluoro-alpha-methyl; CAS RegistryNumber: 5104-494); drugs disclosed in U.S. Pat. Nos. 5,561,161 and5,340,565 (OXiGENE); dexlipotam (Asta Medica); RIP-3 Rigel (Rigel;Pharmaprojects No. 6135); tyloxapol Discovery (Discovery Laboratories;SuperVent; 4-(1,1,3,3-Tetramethylbutyl)phenol polymer with formaldehydeandoxirane; CAS Registry Number: 25301-024); IZP-97001 (Inflazyme);IZP-96005 (Inflazyme); IZP-96002 (Inflazyme); sortac (Inflazyme;IPL-400); BXT-51072 (OXIS; 2H-1,2-Benzoselenazine,3,4-dihydro-4,4-dimethyl-; CAS Registry Number: 173026-17-0); SP-100030(Celgene;2-chloro-N-(3,5-di(trifluoromethyl)phenyl)-4-(trifluo-romethyl)pyrimidine-5-carboxamide);IPL-576092 (Inflazyme; Stigmastan-15-one,22,29-epoxy-3,4,6,7,29-pentahydroxy-, (3alpha, 4beta, 5alpha, 6alpha,7beta, 14beta, 22S); CAS Registry Number: 137571-30-3; U.S. Pat. No.6,046,185); P54 (Phytopharm); Interleukin-10 (Schering-Plough; SCH52000; Tenovil; rI-10; rhIL-10; CAS Registry Number: 149824-15-7); andantisense oligonucleotides PLGA/PEG microparticles.

The NFkB associated polynucleotides and polypeptides of the presentinvention, including agonists, and/or fragments thereof, have uses thatinclude detecting, prognosing, treating, preventing, and/or amelioratingthe following diseases and/or disorders, including, but limited to:immune disorders, inflammatory disorders, aberrant apoptosis, hepaticdisorders, Hodgkins lymphomas, hematopoietic tumors, hyper-IgMsyndromes, hypohydrotic ectodermal dysplasia, X-linked anhidroticectodermal dysplasia, Immunodeficiency, al incontinentia pigmenti, viralinfections, HIV-1, HTLV-1, hepatitis B, hepatitis C, EBV, influenza,viral replication, host cell survival, and evasion of immune responses,rheumatoid arthritis inflammatory bowel disease, colitis, asthma,atherosclerosis, cachexia, euthyroid sick syndrome, stroke, and EAE.

Alternatively, antagonists and/or fragments of the NFkB associatedpolynucleotides and polypeptides of the present invention have uses thatinclude detecting, prognosing, treating, preventing, and/or amelioratingthe following diseases and/or disorders: immune disorders, inflammatorydisorders, aberrant apoptosis, hepatic disorders, Hodgkins lymphomas,hematopoietic tumors, hyper-IgM syndromes, hypohydrotic ectodermaldysplasia, X-linked anhidrotic ectodermal dysplasia, immunodeficiency,al incontinentia pigmenti, viral infections, HIV-1, HTLV-1, hepatitis B,hepatitis C, EBV, influenza, viral replication, host cell survival, andevasion of immune responses, rheumatoid arthritis, inflammatory boweldisease, colitis, asthma, atherosclerosis, cachexia, euthyroid sicksyndrome, stroke, ischemia, ischemia/reperfusion, hypoxia, heartdisease, ischemic diseases of other organs, including, but not limitedto muscle, liver, kidney, the GI tract, cardiac hypertrophy,cardiomyopathy, heart failure, developmental defects of skeletal muscle,cancers of all types and EAE.

In another embodiment, the present invention provides for the use forpolymers to deliver a concatemer to a cell as a complex. In anotherembodiment, the present invention provides for the use for polymers ofthe present invention to deliver a concaternerized double-strandedoligonucleotide molecules (CODN) for transcription factor decoys. In oneembodiment, the concatemers consist of a variable number of end-to-endrepeated copies of a short (having at least 5, 10, 15, 20, 25, 30, 40,45, 50, 75, 100 or more bp) dsDNA containing a sequence or sequencesthat act as transcription factor decoys.

In another embodiment, the present invention provides for the use of thepolymers for covalent addition of targeting peptides, receptor bindingpeptides/protein domains and antibody fragments that may be used totarget the CODN/polymer complexes to a specific cell type; thus theagent can be made organ, tissue and/or cell-type specific.

In another embodiment, the present invention provides for usingpolyamides for targeting peptides and/or antibodies for specific stressand/or drug induced cellular receptors. In one embodiment, thepolyamides target the CODN/polymer complexes to ischemic, inflamed orcancerous tissues.

In another embodiment, the present invention provides for using linkerpeptides containing the sequence recognized by the TNF-alpha convertingenzyme (TACE) or another exopeptidase or endopeptidase in order to allowthe agent to deliver the CODN/polymer complexes to the cell and thencleave off the targeting peptide.

In another embodiment, the present invention provides for using thepolyamides to deliver intact genes (transgenes), plasmids, RNAi, siRNA,morpholinos or other kinds of RNA, proteins and polynucleotides. In oneembodiment, the genes incorporate tissue-specific promoters,controllable promoters, promoters that may be silenced by specificCODN/polymer combinations and may constitute two- and three-unit systemsfor gene expression, control and DNA transposition (i.e. insertion,excision and targeting of transgenes and other DNA molecules).

In another embodiment, the present invention provides for use of thepolyamides in vitro or in vivo, in isolated cells or intact animals inwhich specific blockade of transcription factors or delivery of DNA orother biological effector is desirable. In one embodiment, this includesuse as a research tool, including studies of specific genes and studiesto identify specific genes regulated by the transcription factorstargeted (relates to development of specific CODN/polymer complexes andrelated gene marker mouse lines described below). For clinical use, thiswould include, but is not limited to delivery of transcription factordecoys (e.g. CODNs) that block transcription factors implicated indisease, response to surgery and/or trauma, developmental defects,aging, toxic exposure, etc.

In another embodiment, the present invention provides for transgenicmice expressing marker genes (lacZ and/or GFP variants) under thecontrol of promoter elements that are primarily controlled by specifictranscription factors. In one embodiment, the mice are providedseparately or as a kit including specific CODN/polymer complexes and thematching mouse, which serves to identify the cells in which the markeractivation (experimentally activated) is blocked by the CODN. In anotherembodiment, there are transgenic mice with marker genes that aretranscriptionally turned on, which can be specifically turned off usingCODN/polymer complexes.

In another embodiment, the present invention provides for bi-transgenic(or multiple transgenic) systems designed to utilize the CODN/polymercomplexes to regulate gene expression (up, down, on or off) or tomediate gene transposition (insertion, excision or moving in thegenome). In one embodiment, transgene A may express a gene of interestunder control of a promoter that is inducible by NF-kB or by a yeast orbacterial transcription factor (tetR or Gal4). In one embodiment, thegene would be on after an NF-kb-inducing stimulus, or constitutively onin a tissue expressing the specific transcription factor and the genecould be turned off by simply providing the CODN/polymer complex for thespecific transcription factor (CODN-OFF). In another embodiment, theanimals are continuously delivered CODN/polymer complex and then theCODN/polymer complex is withdrawn to turn the gene on. Other versionscould have the gene off, due to expression in the same cells of atranscriptional repressor (has been described for tet), and therepression reversed by adding CODN/polymer complex, allowing expressionto turn on (CODN-ON).

In another embodiment, the present invention provides for polymersdesigned for variable release/biodegradation; which may be selected forquick degradation/release of CODN, others for long half-life (the CODNmay be active whether or not it is released by the polymers).

1. A method of delivering a biologically active molecule to a cell,comprising contacting the cell with (a) a biologically active moleculeand (b) a cellular delivery polymer.
 2. The method of claim 1, whereinthe cellular delivery polymer is one or more polymers selected from thegroup consisting of polyamides, dendritic macromolecules, andcarbohydrate-containing degradable polyesters.
 3. The method of claim 1,wherein the biologically active molecule is at least one nucleic acidmolecule or at least one polypeptide or both.
 4. The method of claim 1,wherein the polypeptide or the nucleic acid and the cellular deliverypolymer are admixed before the contacting.
 5. The method of claim 4,wherein the cellular delivery polymer forms a polyplex with thebiologically active molecule.
 6. The method of claim 4, wherein thebiologically active molecule is a nucleic acid.
 7. The method of claim6, wherein the nucleic acid is an oligonucleotide.
 8. The method ofclaim 7, wherein the nucleic acid is selected from the group consistingof mRNA, tmRNA, tRNA, rRNA, siRNA, shRNA, PNA, ssRNA, dsRNA, ssDNA,dsDNA, DNA:RNA hybrid molecules, plasmids, artificial chromosomes, genetherapy constructs, cDNA, PCR products, restriction fragments,ribozymes, antisense constructs, and combinations thereof.
 9. A kitcomprising at least one biologically active molecule and at least onecellular delivery polymer.
 10. The kit of claim 9, wherein thebiologically active molecule is a nucleic acid.
 11. The kit of claim 10,wherein the nucleic acid is selected from the group consisting of mRNA,tmRNA, tRNA, rRNA, siRNA, shRNA, PNA, ssRNA, dsRNA, ssDNA, dsDNA,DNA:RNA hybrid molecules, plasmids, artificial chromosomes, gene therapyconstructs, cDNA, PCR products, restriction fragments, ribozymes,antisense constructs, and combinations thereof
 12. A complex comprisinga cellular delivery polymer and an agent that is desirably taken up bycells, wherein the cellular delivery polymer comprises a polycationicpolymer structure.
 13. The complex of claim 12, wherein the deliverypolymer comprises one or more polymers selected from the groupconsisting of polyamides, dendritic macromolecules, andcarbohydrate-containing degradable polyesters.
 14. The complex of claim12, wherein the biologically active molecule is at least one nucleicacid molecule or at least one polypeptide or both.
 15. The complex ofclaim 14, wherein the cellular delivery polymer forms a polyplex withthe biologically active molecule.
 16. The complex of claim 14, whereinthe biologically active molecule is a nucleic acid.
 17. The complex ofclaim 12, wherein the nucleic acid is an oligonucleotide.
 18. Thecomplex of claim 17, wherein the nucleic acid comprises from about 5bases to about 200 kilobases.
 19. The complex of claim 18, wherein thenucleic acid is selected from the group consisting of mRNA, tmRNA, tRNA,rRNA, siRNA, shRNA, PNA, ssRNA, dsRNA, ssDNA, dsDNA, DNA:RNA hybridmolecules, plasmids, artificial chromosomes, gene therapy constructs,cDNA, PCR products, restriction fragments, ribozymes, antisenseconstructs, and combinations thereof.
 20. The complex of claim 19,wherein the nucleic acid comprises one or more chemical modifications.21. The complex of claim 19, wherein the nucleic acid is the agent thatis desirably taken up by cells.
 22. A composition comprising themolecular complex of claim
 12. 23. A cell comprising the molecularcomplex of claim
 12. 24. A composition comprising the cell of claim 23.25. A container comprising the molecular complex of claim
 12. 26. Apharmaceutical composition comprising the molecular complex of claim 12and a pharmaceutically acceptable excipient or carrier.
 27. Thepharmaceutical composition of claim 26, wherein the biologically activemolecule is at least one nucleic acid molecule or at least onepolypeptide or both.
 28. The pharmaceutical composition of claim 27,wherein the biologically active molecule is at least one nucleic acidselected from the group consisting of mRNA, tmRNA, tRNA, rRNA, siRNA,shRNA, PNA, ssRNA, dsRNA, ssDNA, dsDNA, DNA:RNA hybrid molecules,plasmids, artificial chromosomes, gene therapy constructs, cDNA, PCRproducts, restriction fragments, ribozymes, antisense constructs, andcombinations thereof.
 29. The pharmaceutical composition of claim 26,wherein the biologically active molecule is a polypeptide.
 30. A methodof treating an individual suffering from a disease or disorder, themethod comprising contacting the individual with the complex of claim12, the composition of claim 24, or the pharmaceutical composition ofclaim
 26. 31. A method of providing gene therapy to an individual inneed thereof, comprising contacting the individual, or cells therefrom,with the complex of claim 12, the composition of claim 24, or thepharmaceutical composition of claim
 26. 32. The compound according toany one of the preceding claims, wherein said compound is an antisenseoligonucleotide composed of DNA or RNA or an analogue or mimic of DNA orRNA including but not restricted to the following: methylphosphonate,N3′->P5′-phosphoramidate, morpholino, peptide nucleic acid (PNA), lockednucleic acid (LNA), arabinosyl nucleic acid (ANA), fluoro-arabinosylnucleic acid (FANA) methoxy-ethyl nucleic acid (MOE).